JP2014523736A - SUV39H2 as a target gene for cancer treatment and diagnosis - Google Patents

Abstract

Objective method for detecting or diagnosing cancer or determining a predisposition to develop cancer, especially lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor Are described herein. In one embodiment, the diagnostic method comprises determining the expression level of the SUV39H2 gene. The present invention further provides methods for screening candidate substances useful in the treatment and / or prevention of SUV39H2-related cancers such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumors. The present invention further provides a method of inhibiting cell proliferation, thereby treating or alleviating symptoms of SUV39H2-related cancer. The invention also features double-stranded molecules for the SUV39H2 gene and vectors encoding the same, as well as compositions containing such components and their uses related to the treatment and prevention of SUV39H2-related cancers.

Description

  The present invention relates to a method for detecting or diagnosing a predisposition to develop cancer, particularly lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. The present invention also provides candidate substances for the treatment or prevention of cancers associated with overexpression of the SUV39H2 gene, such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. The present invention relates to a screening method. Furthermore, the present invention relates to a double-stranded molecule that inhibits or reduces the expression of the SUV39H2 gene and its therapeutic use.

This application claims the benefit of US Provisional Patent Application No. 61 / 493,235, filed June 3, 2011, the entire contents of which are hereby incorporated by reference.

  A nucleosome is a basic unit of DNA packaging in eukaryotes and a basic unit of chromatin structure consisting of 147 bp of DNA wound around a histone protein core in order (Non-patent Document 1). All four core histones (H3, H4, H2A and H2B) have an unstructured N-terminal tail, and the N-terminus of these histones is subject to a particularly diverse array of post-translational modifications: acetyl , Methylation, phosphorylation, ubiquitination, SUMOylation and ADP-ribosylation (Non-patent Document 2). These histone modifications cause dynamic changes in chromatin structure, thereby affecting transcriptional regulation, DNA replication, DNA repair, and alternative splicing (Non-Patent Documents 3 and 4). Among these epigenetic marks for histones, the methylation process is particularly important for transcriptional regulation (5). Five lysine residues (H3K4, H3K9, H3K27, H3K36 and H4K20) are located in the N-terminal tail and are representative lysines that can be mono-, di-, or trimethylated. While methylation of H3K9, H3K27 and H4K20 mainly suppresses transcription, the methylation marks of H3K4 and H3K36 are related to induction of active transcription (Non-patent Document 6). For example, histone H3 methylation at lysine 9 (H3K9) is one of the most abundant and stable histone modifications and is involved in both gene suppression and heterochromatin formation. H3K9 can be mono-, di-, or trimethylated on H3K9, while the silent intrinsic chromatin region is enriched for mono- and dimethylated H3K9 (Non-Patent Document 17). In mammals, the heterochromatin region is highly trimethylated with H3K9, while the silent intrinsic chromatin region is enriched for mono and dimethylated H3K9 (Non-Patent Document 17). H3K9 methylation is associated with novel gene silencing and DNA methylation and is inherited after mitosis in a coupled manner with DNA methylation.

  Some histone methyltransferases and demethylases have been reported to be deeply involved in human carcinogenesis (Non-Patent Documents 7, 8, 9, 10, 11). For example, SMYD3, PRMT1, PRMT6, SUV420H1 and SUV420H2 have been shown to stimulate cell proliferation through their enzymatic activity (Patent Document 1, Non-Patent Documents 8, 9, 12, 13, 14, 18).

SUV39H2 is also known as KMT1B (Non-Patent Document 15)
A histone methyltransferase containing a SET domain, which is known to methylate H3K9 lysine residues. Suv39h2 is a mouse homologue of human SUV39H2, isolated and characterized as the second mouse Suv39h gene, and has been found to have 59% identity with Suv39h1 (Non-patent Document 16). Expression of Suv39h2 is restricted to the adult testis, and the immunolocalization of the endogenous Suv39h2 protein reveals a heterochromatin-enriched distribution during the first meiotic prophase at the early stage of spermatogenesis . During the middle pachytene phase, Suv39h2 specifically accumulates in the chromatin of silenced sex chromosomes present in the XY body. Furthermore, the histone methyltransferase activity of Suv39h2 appears to play an important role in the regulation of high-level chromatin dynamics during male meiosis (Non-Patent Document 16).
To date, however, there has been no suggestion or proof of the importance of SUV39H2 deregulation in human carcinogenesis.
Cited Reference List Patent Literature

[Patent Document 1] WO2005 / 071102
Non-patent literature

[Non-Patent Document 1]
Strahl BD et al. Nature 2000; 403: 41-45
[Non-Patent Document 2]
Kouzarides T et al. Cell 2007; 128: 693-705
[Non-Patent Document 3]
Huertas D et al. Epigenetics 2009; 4: 31–42
[Non-Patent Document 4]
Luco RF et al. Science 2010; 327: 996-1000
[Non-Patent Document 5]
Kouzarides T et al. Curr Opin Genet Dev 2002; 12: 198-209
[Non-Patent Document 6]
Peterson CL et al. Curr Biol 2004; 14: R546-551
[Non-Patent Document 7]
Cho HS et al. Cancer Res 2010
[Non-Patent Document 8]
Hamamoto R et al. Nat Cell Biol 2004; 6: 731-40
[Non-patent document 9]
Hamamoto R et al. Cancer Sci 2006; 97: 113-8
[Non-Patent Document 10]
Yoshimatsu M et al. Int J Cancer 2011; 128: 562-573
[Non-Patent Document 11]
Hayami S et al. Int J Cancer 2011; 128: 574-586
[Non-patent document 12]
Kunizaki M et al. Cancer Res 2007; 67: 10759-65
[Non-Patent Document 13]
Silva FP et al. Oncogene 2008; 27: 2686-92
[Non-Patent Document 14]
Tsuge M et al. Nat Genet 2005; 37: 1104-7
[Non-Patent Document 15]
Allis CD et al. Cell 2007; 131: 633-636
[Non-Patent Document 16]
O'Carroll D et al. Mol Cell Biol 2000; 20: 9423-9433
[Non-Patent Document 17]
Martin C et al. Nat Rev Mol Cell Biol 2005; 6: 838-49
[Non-Patent Document 18]
Schneider R et al. Trends Biochem Sci 2002; 27: 396-402.

The present invention relates to SUV39H2 and its role in carcinogenesis. Accordingly, the present invention relates to novel compositions and methods for the detection, diagnosis, treatment and / or prevention of cancer, particularly lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. The present invention also relates to a screening method for candidate substances useful for one or both of cancer prevention and treatment.
The heart of the present invention is the discovery that the SUV39H2 gene is overexpressed in cancer cells and that SUV39H2 knockdown by double-stranded molecules against the SUV39H2 gene results in significant suppression of cancer cell growth. Such a double-stranded molecule can be composed of a sense strand and an antisense strand, wherein the sense strand is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 19 and 20 And the antisense strand comprises a sequence complementary to the sense strand, wherein the sense and antisense strands of the molecule hybridize to each other to form a double stranded molecule.

  Accordingly, it is an object of the present invention to provide an isolated double-stranded molecule that, when introduced into a cell, inhibits the expression of the SUV39H2 gene as well as the proliferation of the cell, the molecule comprising a sense strand And the antisense strand complementary thereto, these strands hybridize to each other to form a double-stranded molecule. These double stranded molecules may be utilized in an isolated state, or may be encoded and expressed from a vector. Accordingly, in another aspect, the present invention provides such double-stranded molecules and vectors and host cells that express them.

  Method for inhibiting proliferation of SUV39H2-expressing cells by administering the double-stranded molecule or vector of the present invention to a subject in need thereof and cancer, such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma It is another object of the present invention to provide a method for treating SUV39H2-related diseases such as prostate cancer and soft tissue tumors. Such methods include administering to the subject a composition comprising one or more of the double-stranded molecules or vectors of the invention.

  A further object of the invention is a composition for the treatment or prevention of SUV39H2-related diseases such as cancer, eg lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. And such compositions comprise at least one of the double-stranded molecules or vectors of the invention.

  Still another object of the present invention is to provide a method for detecting or diagnosing cancer in a subject by using the expression level of the SUV39H2 gene as an index. More specifically, an increase in the expression level of a test gene (ie, SUV39H2) in a subject-derived biological sample relative to the normal control level of the test gene is indicative of the presence of cancer in the subject or whether the subject has cancer. Examples of cancers include lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumors.

  Yet another object of the present invention is useful for the treatment and / or prevention of SUV39H2-related diseases such as cancer, eg lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. It is to provide a method for screening candidate substances. Useful candidate substances can be selected using as an index the binding activity against the SUV39H2 polypeptide, the inhibitory activity against the biological activity of the SUV39H2 polypeptide, or the suppressive activity against the expression level of the SUV39H2 gene. Biological activities of SUV39H2 polypeptides that are of particular interest as screening methods of the present invention include cell growth promoting activity and methyltransferase activity (eg, histone methyltransferase activity).

  Yet another object of the present invention is to provide a kit for detecting or diagnosing cancer comprising a reagent for detecting the biological activity of SUV39H2 mRNA, SUV39H2 protein or SUV39H2 protein. A series of specific objectives are described below. Those skilled in the art will appreciate that one or more aspects of the invention may be suitable for one purpose, while one or more other aspects may be suitable for some other purpose. It will be. Each objective may not apply equally to individual aspects of the invention, in all its aspects. As such, the following objectives may be considered as options for any one aspect of this invention.

More specifically, the present invention provides the following [1] to [29]:
[1] A method for detecting or diagnosing cancer in a subject, or for determining a predisposition to develop cancer, comprising a method for determining an expression level of a SUV39H2 gene in a subject-derived biological sample, Wherein an increase in the level compared to a normal control level of the gene indicates that the subject is suffering from or at risk of developing cancer;
Said method wherein said expression level is determined by a method selected from the group consisting of:
(A) a method for detecting mRNA of the SUV39H2 gene;
(B) a method for detecting the protein encoded by the SUV39H2 gene; and (c) a method for determining the biological activity of the protein encoded by the SUV39H2 gene;
[2] The method according to [1], wherein the increase is at least 10% higher than a normal control level;
[3] The method according to [1] above, wherein the subject-derived biological sample is a biopsy specimen, saliva, sputum, blood, serum, plasma, pleural effusion or urine;
[4] A kit for detecting or diagnosing cancer or determining a predisposition to develop cancer, comprising a reagent selected from the group consisting of: (a) a reagent for detecting mRNA of the SUV39H2 gene ;
(B) a reagent for detecting a protein encoded by the SUV39H2 gene;
(C) a reagent for detecting the biological activity of the protein encoded by the SUV39H2 gene;
[5] The kit according to [4], wherein the reagent is a probe for a transcript of the SUV39H2 gene;
[6] The kit according to [4], wherein the reagent is an antibody against a protein encoded by the SUV39H2 gene;
[7] The method according to any one of [1] to [3] above or the above [4] to [6], wherein the biological activity to be detected is cell growth promoting activity or methyltransferase activity. The kit according to any one of the above;
[8] Any one of [1] to [3], wherein the cancer is selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer, and soft tissue tumor. Or the kit according to any one of [4] to [6] above;
[9] A method for screening candidate substances for inhibiting or inhibiting cancer cell growth, or one or both of treating or preventing cancer, comprising the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting a binding activity between the polypeptide or a functional equivalent thereof and a test substance; and (c) selecting a test substance that binds to the polypeptide or a functional equivalent thereof;
[10] A screening method for a candidate substance for inhibiting or inhibiting cancer cell growth, or one or both of treating or preventing cancer, comprising the following steps:
(A) contacting the test substance with cells expressing the SUV39H2 gene; and (b) selecting a test substance that reduces the expression level of the SUV39H2 gene compared to the expression level in the absence of the test substance;
[11] A screening method for a candidate substance for inhibiting or inhibiting cancer cell growth, or one or both of cancer treatment or prevention, comprising the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting the biological activity of the polypeptide of step (a) or a functional equivalent thereof;
(C) selecting a test substance that inhibits the biological activity of the polypeptide or a functional equivalent thereof compared to the biological activity detected in the absence of the test substance;
[12] The method according to [11] above, wherein the biological activity is cell growth promoting activity or methyltransferase activity;
[13] A method for screening a candidate substance for inhibiting or inhibiting cancer cell growth, or one or both of treating or preventing cancer, comprising the following steps:
(A) contacting a test substance with a cell into which a vector containing a transcription regulatory region of the SUV39H2 gene and a reporter gene expressed under the control of the transcription regulatory region has been introduced;
(B) measuring the expression or activity level of the reporter gene; and (c) selecting a test substance that reduces the expression activity or level of the reporter gene compared to the level in the absence of the test substance. ;
[14] A screening method for a candidate substance for inhibiting cancer cell growth or one or both of cancer treatment or prevention, comprising the following steps:
(A) contacting the test substance with a cell expressing a SUV39H2 gene and a downstream gene selected from the genes shown in Tables 7 and 8;
(B) detecting the expression level of the downstream gene;
(C) selecting a test substance that changes the expression level of the downstream gene compared to the expression level detected in the absence of the test substance;
[15] The downstream gene of the SUV39H2 gene is selected from the genes shown in Table 7, and the expression of the downstream gene is compared with the expression level detected in the step (c) in the absence of the test substance. The method according to [14] above, which is a step of selecting a test substance that increases the level;
[16] The downstream gene of the SUV39H2 gene is selected from the genes shown in Table 8, and the expression of the downstream gene is compared with the expression level detected in the step (c) in the absence of the test substance. The method according to [14] above, which is a step of selecting a test substance that reduces the level;
[17] The cancer according to any one of [9] to [16], wherein the cancer is selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer, and soft tissue tumor. the method of;
[18] A double-stranded molecule comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, and the antisense strand comprising the target strand A nucleotide sequence complementary to the sequence, wherein the sense strand and the antisense strand hybridize to each other to form a double-stranded molecule, which is introduced into a cell expressing the SUV39H2 gene. A double-stranded molecule that inhibits the expression of the SUV39H2 gene if
[19] The double-stranded molecule according to the above [18], wherein the double-stranded molecule is about 19 to about 25 nucleotides in length;
[20] The double-stranded molecule according to [18], wherein the double-stranded molecule is a single polynucleotide molecule comprising a sense strand and an antisense strand linked via a single-stranded nucleotide sequence;
[21] The double-stranded molecule has the general formula:
5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′
Wherein [A] is a sense strand comprising a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, [B] is a nucleotide sequence comprising about 3 to about 23 nucleotides, and [A ′] is the target sequence. The double-stranded molecule according to the above [20], which is an antisense strand comprising a complementary nucleotide sequence;
[22] A vector encoding the double-stranded molecule according to any one of [18] to [21];
[23] A vector comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises the nucleotide sequence of SEQ ID NO: 19 or 20, and the antisense strand nucleic acid is a nucleotide complementary to the sense strand A vector composed of a sequence, wherein the transcript of the sense strand and the antisense strand hybridizes to each other to form a double-stranded molecule, and the vector inhibits the expression of the SUV39H2 gene;
[24] One or both methods of treating or preventing cancer in a subject, the method encoding the subject with a pharmaceutically effective amount of a double-stranded molecule against the SUV39H2 gene or the double-stranded molecule Administering a vector, wherein the double-stranded molecule inhibits SUV39H2 gene expression as well as cell proliferation when introduced into a cell expressing the SUV39H2 gene;
[25] The method according to [24], wherein the double-stranded molecule is the double-stranded molecule according to any one of [18] to [21];
[26] The method described in [24] above, wherein the vector is the vector described in [22] or [23] above;
[27] A composition for one or both of the treatment or prevention of cancer, the composition comprising a pharmaceutically effective amount of a double-stranded molecule against the SUV39H2 gene or a vector encoding the double-stranded molecule and A composition comprising a pharmaceutically acceptable carrier, wherein said double-stranded molecule inhibits expression of SUV39H2 gene as well as cell proliferation when introduced into a cell expressing SUV39H2 gene;
[28] The composition according to [27], wherein the double-stranded molecule is the double-stranded molecule according to any one of [18] to [21];
[29] The composition described in [27] above, wherein the vector is the vector described in [22] or [23].

  These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of the preferred embodiments and are not limiting of the invention or other alternative embodiments of the invention. It is. Other objects and features of the present invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. In particular, while the present invention has been described herein with reference to a number of specific embodiments, the description is illustrative of the invention and is not to be construed as limiting the invention. Will be understood. Various modifications and applications can occur to those skilled in the art without departing from the spirit and scope of the invention as set forth in the appended claims. Similarly, other objects, features, effectiveness and advantages of the present invention will be apparent from this summary and certain embodiments described below and will be readily apparent to those skilled in the art. Such objects, features, effectiveness and advantages can be found in the above, alone or in the references incorporated herein, along with the accompanying examples, data, drawings and all reasonable inferences derived from them. It will be clear in consideration.

Various aspects and applications of the present invention will become apparent to those skilled in the art in view of the following brief description of the drawings and the detailed description and preferred embodiments of the present invention:
FIG. 1 shows SUV39H2 overexpression in clinical lung cancer. Part A shows SUV39H2 mRNA levels in 14 lung cancer cases (NSCLC: 9 cases, SCLC: 5 cases) and 16 normal vital organs. Part B represents quantitative real-time PCR measurements using 14 lung cancer samples and 14 normal tissues, and the results are shown by a box diagram. For statistical analysis, Kruskal-Wallis test (* P <0.05) and Student's t-test (** P <0.05) were performed. Part C represents SUV39H2 immunohistochemical staining in normal adult human tissue. All tissue samples were purchased from BioChain. Magnification: x200.

FIG. 2 reveals elevated expression of SUV39H2 in lung cancer tissue. 1 shows a tissue microarray image of a lung tumor stained by standard immunohistochemical analysis for SUV39H2 protein. Immunohistochemical analysis of SUV39H2 protein expression in lung cancer tissue indicates that SUV39H2 is abundantly expressed in the nucleus. Clinical information for each section is represented on the histology. All tissue samples were purchased from BioChain. Magnification: x200.

FIG. 3 reveals significant upregulation of SUV39H2 in bladder cancer samples. Part A represents quantitative real-time PCR measurements using 125 bladder cancer tissues and 28 normal bladder samples (British), and the results are shown in a box plot. For statistical analysis, Student's t-test was adopted (* P <0.05). Part B represents a comparison of SUV39H2 expression between normal and tumor bladder tissue in Japanese patients. The signal intensity for each sample is analyzed by cDNA microarray, and the results are shown as box diagrams (median value (50%) is boxed). The Mann-Whitney's U-test was used for statistical analysis. Part C represents an immunohistochemical analysis showing upregulation of SUV39H2 in bladder cancer and normal bladder tissue purchased from BioChain. Clinical information for each section is represented on the histology. All tissue samples were purchased from BioChain. Magnification: x200.

FIG. 4 shows the growth inhibitory effect of SUV39H2 knockdown. Part A represents SUV39H2 mRNA expression levels in various cell lines. Quantitative real-time PCR was performed using normal cell lines (IMR-90, WI-38, SAEC and CCD-18Co), three lung cancer cell lines (A549, RERF and SBC-5) and three bladder cancer cell lines (SW780, RT4 And SCaBER). Part B represents verification of SUV39H2 protein expression levels in various cell lines. Lysates from 3 normal cell lines (IMR-90, WI-38 and SAEC) and 3 lung cancer cell lines (A549, RERF and SBC5) were immunoblotted with anti-SUV39H2 antibody. ACTB was used as an internal control. Part C represents the results of quantitative real-time PCR showing suppression of endogenous SUV39H2 expression by two independent SUV39H2-specific siRNAs (siSUV39H2 # 1, # 2) in A549 and SW780 cells. siRNA targeting EGFP (siEGFP) and siNegative control (siNC) were used as controls. mRNA expression levels are normalized by GAPDH expression and values are relative to siEGFP (siEGFP = 1). Results are the mean ± SD of 3 independent experiments. Part D represents the effect of SUV39H2 knockdown on the growth of cancer cell lines (A549 and SW780) and normal cell lines (CCD-18Co). The cell number was measured by the cell counting kit 8. Relative cell numbers were normalized to the number of siEGFP treated cells (siEGFP = 1): results are the mean ± SD of 3 independent experiments. P values were calculated using Student's t-test (*, P <0.05). Parts E and F represent the results of a colony formation assay to confirm the effect of SUV39H2 expression on cell growth regulation. Part E represents loss of function experiments with SUV39H2 siRNA. Giemsa staining was performed 9 days after treatment with siRNA (A549) and 3 days after (SW780).

Part F represents the results of the SUV39H2 clonogenicity assay. The methylation activity of SUV39H2 is important for its growth promoting effect. COS7 cells were transfected with FLAG-Mock, -SUV39H2 (WT or Δ-SET) and stained with Giemsa 14 days after transfection.

FIG. 5 shows the effect of inhibition of SUV39H2 expression on cell cycle distribution. Part A shows the effect of SUV39H2 knockdown on cell cycle dynamics in cancer cells. A549 and SW780 cells were treated with siRNA and analyzed by FACS 72 hours after siRNA treatment. A representative histogram of this experiment is shown. Numerical analysis of FACS results, cell classification by cell cycle status. Mean ± SD of 3 independent experiments. P values were calculated using Student's t-test. Part B represents a histogram showing the percentage of cells in each cycle shown in Part A.

FIG. 6 shows overexpression of SUV39H2 in lung cancer cells relative to a normal cell line (human small airway epithelial cells: SAEC). The expression level of SUV39H2 was analyzed by quantitative real-time PCR. Relative SUV39H2 expression indicates the ratio compared to the value in SAEC. ADC: Adenocarcinoma; SCC: Squamous cell carcinoma; LCC: Large cell carcinoma; SCLC: Small cell lung cancer.

FIG. 7 shows that SUV39H2 is important for cancer cell growth. Part A represents the results of a colony formation assay of SBC5 cells. Giemsa staining was performed 10 days after siRNA treatment. Part B represents the delayed cell growth of Suv39hDNMEFs compared to Suv39hWT. 1 × 10 ⁵ cells of Suv39hWT and Suv39hDN (SUV39H1 and SUV39H2 double null) MEFs were seeded in 6-well tissue culture plates and cell proliferation assays were performed according to the time courses indicated above.

Part C represents the cell cycle distribution of Suv39WT and Suv39DN mouse embryonic fibroblasts (MEFs). Suv39WT and Suv39DN MEFs were seeded in 6-well tissue culture plates for 72 hours and fluorescein isothiocyanate (FITC) conjugated anti-BrdU and 7-amino-actinomycin D (7-AAD) as described in Materials and Methods. The cell cycle distribution was analyzed by flow cytometry after coupling staining with A representative histogram of the DN to WT ratio at each cell stage is shown.

FIG. 8 displays the results of a two-dimensional, non-hierarchical clustering analysis of SW780 and A549 mRNA expression profiles after knockdown of SUV39H2 expression. Differentially expressed genes were selected for this analysis. The genes that are up-regulated are shown in Table 7. The genes that are down-regulated are shown in Table 8.

FIG. 8B is a continuation of FIG. 8A.

FIG. 9 shows the expression level of SUV39H2 in 78 normal tissues. Data were obtained from BioGPS (http://biogps.gnf.org/#goto=genereport&id=79723). GAPDH expression is shown as a control for signal intensity. The fact that almost no bar corresponding to SUV39H2 is visible confirms that the expression level of SUV39H2 is significantly lower compared to GAPDH in these normal tissues.

FIG. 10 reveals representative examples of positive SUV39H2 expression in lung ADC, SCC tissue and normal lung tissue. Magnification, x100. Aspect description

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of aspects of the present invention, the preferred methods, devices, and materials are now described. However, before describing the materials and methods of the present invention, the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein may be altered according to routine experimentation and optimization. Therefore, it should be understood that the present invention is not limited thereto. The terminology used herein is for the purpose of describing particular types or embodiments only and is not intended to limit the scope of the invention which is limited only by the appended claims. It should also be understood that the disclosure of each publication, patent or patent application referred to herein is specifically incorporated by reference in its entirety. However, nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definition:
As used herein, the words “a”, “an”, and “the” mean “at least one” unless stated otherwise.
The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. The term refers to, in addition to natural amino acid polymers, amino acids in which one or more amino acid residues are modified residues or are non-natural residues, eg, artificial chemical mimetics of the corresponding natural amino acids. Applied to polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Natural amino acids are natural amino acids encoded by the genetic code and natural amino acids that are post-translationally modified in cells (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The phrase “amino acid analog” refers to a compound that has the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and α-carbon attached to an R group), but has a modified R group or modified backbone ( For example, homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). The phrase “amino acid mimetic” refers to a chemical substance that has a different structure but a function similar to that of a general amino acid.
Amino acids are referred to herein as the IUPAC-IUB Biochemical Nomenclature Committee (Biochemical
Nomenclature Commission) can be used in the known three-letter notation or one-letter notation.

  The terms “gene”, “polynucleotide”, “oligonucleotide”, “nucleic acid”, and “nucleic acid molecule” are used interchangeably unless otherwise specified, and are generally accepted single letter codes as well as amino acids. Called by. Like amino acids, they encompass both natural and non-natural nucleic acid polymers. A polynucleotide, oligonucleotide, nucleotide, nucleic acid, or nucleic acid molecule may be composed of DNA, RNA, or a combination thereof.

  In the context of the present invention, the phrase “SUV39H2 gene” encompasses a polynucleotide that encodes either the human SUV39H2 gene or a functional equivalent of the human SUV39H2 gene. In the context of the present invention, the SUV39H2 gene or functional equivalent thereof can be obtained from nature as a natural protein via conventional cloning methods or through chemical synthesis based on selected nucleotide sequences. Methods for cloning genes using cDNA libraries and they are well known in the art.

  The term “isolated” or “purified” as used with respect to a substance (eg, polypeptide, antibody, polynucleotide, etc.) is used if the substance is in its original environment (eg, natural). It is taken from the natural environment) and is thus altered from its natural state. Examples of isolated nucleic acids include DNA (eg, cDNA), RNA (eg, mRNA), and other cellular material or, if produced by recombinant techniques, substantially free of media or chemically synthesized. In this case, derivatives thereof which are substantially free of chemical precursors or other chemical substances are mentioned. In a preferred embodiment, the nucleic acid molecule encoding the antibody of the present invention is isolated or purified.

  An “isolated” or “purified” antibody is substantially free of cellular material, eg, other contaminating proteins from the cell or tissue from which the carbohydrate, lipid or protein (antibody) is derived. Refers to an antibody that is substantially free of chemical precursors or other chemicals when chemically synthesized. The term “substantially free of cellular material” includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cell from which it was isolated or recombinantly produced.

  Thus, a polypeptide that is substantially free of cellular material has less than about 30%, 20%, 10%, or 5% (per dry weight) of a heterologous protein (also referred to herein as “contaminating protein”). Polypeptide preparations are included. When the polypeptide is made recombinantly, in some embodiments, it is also substantially free of culture medium, which contains less than about 20%, 10%, or 5% culture medium of the protein preparation volume. Including preparations of the polypeptide. If the polypeptide is made by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, which are about 30%, 20%, 10% of the volume of the protein preparation. %, Less than 5% (dry weight) of a preparation of polypeptides containing chemical precursors or other chemicals involved in protein synthesis. The inclusion of an isolated or purified polypeptide in a particular protein preparation may include, for example, sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation and subsequent Coomassie brilliant blue staining of the gel. It can be indicated by the appearance of a later single band.

As used herein, the term “biological sample” refers to a whole organism or a subset of its tissues, cells or components (eg, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic fluid umbilical cord) (Amniotic cord) refers to body fluids including but not limited to blood, urine, vaginal fluid and semen. A “biological sample” is further a homogenate, lysate, extract, cell culture or tissue prepared from the whole organism, or a subset of its cells, tissues or components, or a fraction or part thereof. Refers to a culture. Finally, a “biological sample” refers to a nutrient broth or gel containing a cell component (eg, protein or polynucleotide) in which the organism is grown, eg, an organism. In the context of the present invention, the biological sample may comprise tissue or cells, preferably extracted from the lung, neck, bladder, esophagus, or prostate or osteosarcoma or soft tissue tumor.
Unless otherwise specified, the term “cancer” refers to a cancer that overexpresses the SUV39H2 gene. Cancers that overexpress the SUV39H2 gene include, but are not limited to, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer, and soft tissue tumor.

  As long as the methods and compositions of the invention are useful in the context of “prevention and prophylaxis”, these terms are used interchangeably herein, and any term that reduces mortality or morbidity due to disease. Refers to activity. Prevention and prevention can occur at “primary, secondary, and tertiary prevention levels”. Primary prevention and prophylaxis avoid the onset of disease, while secondary and tertiary level prevention (prevention and prophylaxis), along with prevention of disease progression and appearance of symptoms (prevention and prophylaxis) Includes activities aimed at reducing the negative impact of previously established diseases by recovering and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at reducing the severity of certain disorders, eg, reducing tumor growth and metastasis.

  Insofar as certain embodiments of the invention include cancer treatment and / or prevention and / or prevention of post-operative recurrence, such methods may include any of the following steps: Surgical removal of cancer cells, inhibition of cancerous cell growth, tumor regression or regression, suppression of cancer appearance and induction of remission, tumor regression, and reduction or inhibition of metastasis. Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, reduces levels of blood tumor markers, and detects detectable symptoms associated with cancer. ease. Treatment also qualifies as “effective” if it provides clinical efficacy, eg, reduced expression of the SUV39H2 gene or reduced cancer size, spread, or metastatic potential in a subject. Can do. When the treatment is applied prophylactically, “effective” means that the formation of cancer is delayed or inhibited, or the clinical symptoms of cancer are prevented or alleviated. Efficacy is determined in connection with any known diagnosis or treatment method for a particular tumor type.

Genes and polypeptides:
The present invention is based, at least in part, on the discovery that the gene encoding SUV39H2 is overexpressed in some cancers compared to non-cancerous tissues. SUV39H2 (the suppressor of variegation 3-9 homolog 2) is also called KMT1B (Allis CD, et al, Cell 2007; 131: 633-636). It is a transferase.

  Exemplary nucleic acid sequences of the SUV39H2 gene are SEQ ID NO: 21 (GenBank accession No. NM_001193424.1), 23 (GenBank accession No. NM_001193425.1), 25 (GenBank accession No. NM_001193426.1), 27 (GenBank accession No. NM_001193427.1) and 29 (GenBank accession No. NM_024670.3). Exemplary amino acid sequences of polypeptides encoded by the SUV39H2 gene are SEQ ID NO: 22 (GenBank accession No. NP_001180353.1), 24 (GenBank accession No. NP_001180354.1 or NP_078946.1), 26 (GenBank accession No. NP_001180355.1), 28 (GenBank accession No. NP_001180356.1), and 30 (GenBank accession No. NP_078946.1). However, the present invention is not limited to these sequences.

Here, the polypeptide encoded by the SUV39H2 gene is referred to as “SUV39H2 polypeptide” or “SUV39H2 protein” or simply “SUV39H2”. In the context of the present invention, the phrase “SUV39H2 gene” includes not only polynucleotides encoding human SUV39H2 polypeptides but also polynucleotides encoding functional equivalents of the human SUV39H2 gene. The SUV39H2 gene can be obtained from nature as a natural polynucleotide through conventional cloning methods or through chemical synthesis based on a selected nucleotide sequence. As described above and as discussed in detail below, gene cloning methods and the like using cDNA libraries are well known in the art. As mentioned above, the present invention extends to “functional equivalents” of proteins (polypeptides) and these are further examples of “SUV39H2 polypeptides”. As used herein, a “functional equivalent” of a protein (eg, SUV39H2 polypeptide) is a polypeptide having a biological activity equivalent to the original reference protein.
Accordingly, any polypeptide that retains the biological activity of the SUV39H2 protein is considered to be its functional equivalent in the context of the present invention. For example, a functional equivalent of a SUV39H2 polypeptide may retain the cell growth promoting activity and / or methyltransferase activity of a native SUV39H2 polypeptide. In the context of the present invention, functional equivalents of SUV39H2 polypeptides include polymorphic variants, interspecies homologs, and those encoded by alleles of these polypeptides.

  Preferred functional equivalents of SUV39H2 polypeptides retain one or both of the cell growth promoting and methyltransferase activities of the native SUV39H2 polypeptide. A functional equivalent of a SUV39H2 polypeptide that retains the methyltransferase activity of the SUV39H2 polypeptide is expected to retain the SET-domain of the native SUV39H2 polypeptide. In the aforementioned sequence, the SET-domain has amino acid positions 251 to 372 of the amino acid sequence shown in SEQ ID NO: 22, amino acid positions 191 to 312 of the amino acid sequence shown in SEQ ID NO: 24, and amino acid positions 100 to 100 of the amino acid sequence shown in SEQ ID NO: 26. 192, amino acid positions 40 to 132 of the amino acid sequence shown in SEQ ID NO: 28, and 191 to 312 of the amino acid sequence shown in SEQ ID NO: 30. An example of such a functional equivalent is set forth in the amino acid sequence of SEQ ID NO: 32.

  Examples of functional equivalents are those in which one or more, for example, 1-5 amino acids or up to 5% of the original amino acids have been substituted, deleted, added or inserted into the natural amino acid sequence of the SUV39H2 protein. Can be mentioned. Alternatively, the polypeptides are at least about 80% homologous (also referred to as sequence identity) to the sequence of the respective protein, more preferably at least about 90% to 95% homology, often about 96%, 97%, 98 % Or 99% homology amino acid sequence. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the native nucleotide sequence of the SUV39H2 gene.

  The polypeptides described herein may vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, or form depending on the cell or host used to produce them or the purification method used. Also good. Nevertheless, it is within the functional equivalent of SUV39H2 polypeptide as long as it is functionally equivalent to human SUV39H2 protein.

A functional equivalent consisting of a mutated or modified form of a native SUV39H2 polypeptide in which one or more amino acids have been substituted, deleted, added or inserted into the native sequence. It is generally known that modifications of two, two or more amino acids will not significantly impact or affect the function of the protein. In some cases, it can even enhance the desired function of the original protein. Indeed, a mutated or modified protein, ie a protein having an amino acid sequence that has been altered by substitution, deletion, insertion and / or addition of one, two or more amino acid residues of an amino acid sequence, Can retain biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10: 6487-500 (1982); Dalbadie-McFarland
et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)). Thus, those skilled in the art will recognize individual additions or deletions to the amino acid sequence that alter a single amino acid or a low percentage of amino acids (ie, less than 5%, more preferably less than 3%, even more preferably less than 1%). It is recognized that insertions and substitutions, or what are considered “conservative modifications”, where alteration of the protein results in a protein with a similar function is acceptable in the context of the present invention. I will. Thus, a functional equivalent of a SUV39H2 polypeptide may have an amino acid sequence in which one, two or more amino acids have been added, inserted, deleted and / or substituted in the native human SUV39H2 polypeptide sequence. .

  As long as the activity of the SUV39H2 protein is maintained, the site and number of amino acid mutations are not particularly limited. Thus, mutations / modifications (ie, additions, deletions, insertions or substitutions to the amino acid sequence) may be introduced at the N-terminus and C-terminus, as well as at intermediate sites. However, it is generally preferred that no more than 5% of the amino acid sequence is altered, more preferably less than 3%, even more preferably less than 1%. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such variants is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 6 amino acids or less, and even More preferably, it is 3 amino acids or less.

The amino acid residue to be mutated is preferably mutated to a different amino acid that preserves the properties of the amino acid side chain (a process known as conservative amino acid substitution). Examples of amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T) and side chains having the following functional groups or characteristics in common: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S , T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); Side chain (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for each other:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), lysine (K);
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) Phenylalanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, eg, Creighton, Proteins 1984).

  Such conservatively modified polypeptides are encompassed within the functional equivalent of SUV39H2 protein in the context of the present invention. However, the invention is not limited thereto, and functional equivalents of SUV39H2 protein can include non-conservative modifications as long as the resulting modified peptide retains at least one biological activity of the SUV39H2 protein. The number of amino acids to be mutated in such a modified polypeptide is generally 20 amino acids or less, preferably 10 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, or 2 amino acids or less. . Furthermore, variant proteins do not exclude polymorphic variants, interspecies homologs, and those encoded by these alleles.

  An example of a functional equivalent of a SUV39H2 polypeptide modified by the addition of several amino acid residues is a fusion protein of the SUV39H2 polypeptide and other polypeptides or peptides. Such a fusion protein can be ligated by a technique well known to those skilled in the art, for example, by ligating DNA encoding the SUV39H2 gene in such a manner that the DNA encoding the other polypeptide or peptide coincides with the frame and expressing the fusion DNA. And can be produced by expressing it in a host. The “other” component of the fusion protein is typically a small epitope composed of a few to a dozen amino acids. There are no restrictions on the polypeptide or peptide fused to the SUV39H2 polypeptide, so long as the resulting fusion protein retains any one of the desired biological activities of the SUV39H2 polypeptide.

Exemplary fusion proteins contemplated by the present invention include SUV39H2 polypeptides and other small peptides such as FLAG (Hopp TP, et al.,
Biotechnology 6: 1204-10 (1988)), 6xHis containing 10 His (histidine) residues, 10xHis, influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7- Examples include fusions with tags, HSV-tags, E-tags, SV40T antigen fragments, lck tags, α-tubulin fragments, B-tags, protein C fragments and the like. Examples of proteins that can be fused to SUV39H2 polypeptide include GST (glutathione-S-transferase), influenza agglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein), etc. It is.
Furthermore, functional equivalents of SUV39H2 polypeptides do not exclude polymorphic variants, interspecies homologs, and those encoded by alleles of these polypeptides.

Methods known in the art for isolating functional equivalents include:
For example, hybridization techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Lab. Press, 2001) are known. Those skilled in the art will recognize DNA having high homology (ie, sequence identity) with all or part of a human SUV39H2 DNA sequence (eg, SEQ ID NO: 21, 23, 25, 27, or 29) encoding a human SUV39H2 polypeptide. It can be easily isolated and the functional equivalent of the human SUV39H2 polypeptide can be isolated from the isolated DNA.

  Hybridization conditions suitable for isolating DNA encoding a functional equivalent of the human SUV39H2 gene can be routinely selected by those skilled in the art. As used herein, the phrase “stringent conditions” refers to a nucleic acid molecule that hybridizes to its target sequence, typically in a complex mixture of nucleic acids, but to other sequences. In contrast, it refers to conditions that will not detectably hybridize. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidelines for nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of Hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes with the target sequence in equilibrium (the target sequence is present in excess, so , 50% of the probes are occupied in equilibrium). Stringent conditions may be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice background, preferably 10 times background hybridization. Examples of stringent hybridization conditions include: 50% formamide, 5 × SSC, and 1% SDS, 42 ° C. incubation, or 5 × SSC, 1% SDS, 65 ° C. Wash at 50 ° C. in 0.2 × SSC and 0.1% SDS.

In the context of the present invention, the optimal hybridization conditions for isolating DNA encoding a polypeptide functionally equivalent to the human SUV39H2 protein can be routinely selected by those skilled in the art. For example, hybridization is performed by performing prehybridization for 30 minutes or more at 68 ° C. using “Rapid-hyb buffer” (Amersham LIFE SCIENCE), adding a labeled probe, and heating at 68 ° C. for 1 hour or more. May be. The subsequent washing step can be performed, for example, under low stringent conditions. Examples of low stringency conditions include 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. High stringency conditions are often preferred. Exemplary high stringency conditions include 2 × SSC, 0.01% SDS, 3 × 20 minutes at room temperature, followed by 1 × SSC, 0.1% SDS, 3 × 20 minutes at 37 ° C. And washing twice in 1 × SSC, 0.1% SDS at 50 ° C. for 20 minutes. However, several factors, such as temperature and salt concentration, can affect the stringency of hybridization, and those skilled in the art can routinely adjust these and other factors to reach the desired stringency. Can do.
Accordingly, functional equivalents of SUV39H2 polypeptides used in the present invention include polypeptides encoded by DNA that hybridizes under stringent conditions with all or part of the DNA sequence encoding human SUV39H2 polypeptide. Is included. These functional equivalents include mammalian homologues corresponding to human SUV39H2 polypeptides (eg, polypeptides encoded by monkey, mouse, rat, rabbit or bovine SUV39H2 genes).

  Instead of hybridization, DNA (SEQ ID NO: 21, 23) encoding a human SUV39H2 polypeptide (SEQ ID NO: 22, 24, 26, 28 or 30) is used using a gene amplification method, such as the polymerase chain reaction (PCR) method. , 25, 27, or 29), a DNA that encodes a functional equivalent of a human SUV39H2 polypeptide can be isolated using a primer synthesized based on the sequence information. Examples of primer sequences are shown in the semi-quantitative RT-PCR of the examples.

  The functional equivalent of a human SUV39H2 polypeptide encoded by DNA isolated through the hybridization or gene amplification techniques described above is usually highly homologous to the amino acid sequence of the human SUV39H2 polypeptide (also referred to as sequence identity). Called). “High homology” (also referred to as “high sequence identity”) typically refers to the degree of identity between two optimally aligned sequences (either polypeptide or polynucleotide sequences). Typically, high (or) homology or sequence identity is 40% or more, such as 60% or more, such as 80% or more, such as 85%, 90%, 95%, 98%, 99%, or more Refers to higher homology. The degree of homology or identity between two polypeptide or polynucleotide sequences can be determined according to the following algorithm [Wilbur WJ & Lipman DJ. Proc Natl Acad Sci US A. 1983 Feb; 80 (3) : 726-30].

  The percentage of sequence identity and sequence similarity can be readily determined by conventional techniques such as the BLAST and BLAST 2.0 algorithms described [Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3): 403-10; Nucleic Acids Res. 1997 Sep 1; 25 (17): 3389-402]. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov/). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short phrases of length W in the query sequence. This aligns with or satisfies some positive score threshold T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word matches serve as the basis for initiating searches to find longer HSPs containing them.

  This phrase match is then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for matching residue pairs; always> 0) and N (penalty score for mismatched residues; always <0) for nucleotide sequences. For amino acid sequences, a cumulative score is calculated using a scoring matrix. The extension of word match in each direction is when the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of one or more negative score residue alignments Or stop when it reaches the end of either sequence.

  The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default 28 phrase sizes (W), 10 expectations (E), M = 1, N = -2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, 3 phrase sizes (W), 10 extractions (E), and a BLOSUM62 scoring matrix [Henikoff S & Henikoff JG. Proc Natl Acad Sci US A. 1992 Nov 15; 89 (22): 10915-9].

  The invention also includes partial peptides of SUV39H2 polypeptides and their use in screening methods. The partial peptide of the SUV39H2 polypeptide has an amino acid sequence specific for the SUV39H2 polypeptide, preferably less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least 7 amino acids, Preferably, it is composed of 8 amino acids or more, 9 amino acids or more, 10 amino acids or more, 15 amino acids or more, 20 amino acids or more, 50 amino acids or more, or 80 amino acids or more.

The SUV39H2 polypeptides and functional equivalents used in the present invention can be obtained from nature as natural proteins through conventional purification methods, or through chemical synthesis based on selected amino acid sequences. For example, conventional peptide synthesis methods that can be employed for synthesis include the following:
(1) Peptide Synthesis, Interscience, New York, 1966;
(2) The Proteins, Vol. 2, Academic Press, New York, 1976;
(3) Peptide synthesis, Maruzen, 1975;
(4) Fundamentals and experiments of peptide synthesis, Maruzen, 1985;
(5) Development of pharmaceuticals (continued Vol. 14) Peptide synthesis, Hirokawa Shoten, 1991;
(6) WO99 / 67288; and (7) Barany G. & Merrifield RB, Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

  Alternatively, SUV39H2 polypeptides and functional equivalents thereof can be obtained by any known genetic engineering method for making polypeptides (eg, Morrison DA., Et al., J Bacteriol. 1977 Oct; 132 (1): 349-51; Clark-Curtiss JE & Curtiss R 3rd. Methods Enzymol. 1983; 101: 347-62). For example, first a suitable vector containing a polynucleotide encoding a polypeptide of interest in an expressible form (eg, downstream of a regulatory sequence containing a promoter) is prepared to transform a suitable host cell, and The host cell is then cultured to produce the polypeptide. More specifically, the gene encoding the SUV39H2 polypeptide or functional equivalent thereof is inserted into a vector for expressing a foreign gene, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. By doing so, it is expressed in a host (eg, animal) cell or the like.

  In addition to the protein of interest, the vector may also contain a promoter for inducing protein expression. Any commonly used promoter can be used, such as the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, 1982, 83-141), EF-α Promoter (Kim DW, et al. Gene. 1990 Jul 16; 91 (2): 217-23), CAG promoter (Niwa H, et al., Gene. 1991 Dec 15; 108 (2): 193-9), RSV LTR promoter (Cullen BR. Methods Enzymol. 1987; 152: 684-704), SR α promoter (Takebe Y, et al., Mol Cell Biol. 1988 Jan; 8 (1): 466-72), CMV early phase Promoter (Seed B & Aruffo A. Proc Natl Acad Sci US A. 1987 May; 84 (10): 3365-9), SV40 late promoter (Gheysen D & Fiers W. J Mol Appl Genet. 1982; 1 (5): 385-94), adenovirus late promoter (Kaufman RJ, et al., Mol Cell Biol. 1989 Mar; 9 (3): 946-5), H V TK promoter, and the like.

Introduction of a vector into a host cell for expressing a gene encoding a SUV39H2 polypeptide or a functional equivalent thereof can be performed according to any method, for example, electroporation (Chu G, et al. Nucleic Acids Res. 1987 Feb 11; 15 (3): 1311-26), calcium phosphate method (Chen C & Okayama H. Mol Cell Biol. 1987 Aug; 7 (8): 2745-52), DEAE-dextran method ( Lopata MA, et al., Nucleic Acids Res. 1984 Jul 25; 12 (14): 5707-17; Sussman DJ & Milman G. Mol Cell Biol. 1984 Aug; 4 (8): 1641-3), Lipofectamine method ( Derijard B, et al., Cell. 1994 Mar 25; 76 (6): 1025-37; Lamb BT, et al., Nat Genet. 1993 Sep; 5 (1): 22-30; Rabindran SK, et al. Science. 1993 Jan 8; 259 (5092): 230-4), and the like.
SUV39H2 polypeptides and functional equivalents thereof can also be generated in vitro using an in vitro translation system.

Methods for diagnosing or detecting cancer:
The present invention relates to the discovery that SUV39H2 can serve as a diagnostic marker for cancer and thus finds use in detecting cancers associated therewith. As revealed herein, SUV39H2 gene expression is unique in lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumors (FIGS. 1, 3, 6, 10 and Table 6). Increase significantly and significantly. Thus, the SUV39H2 gene identified here, and its transcription and translation products, find diagnostic utility as a marker for the above cancers and by measuring the expression of the SUV39H2 gene in a sample. . These cancers can be diagnosed or detected by comparing the expression level of the SUV39H2 gene in the subject-derived sample with that in a normal sample. Specifically, the present invention provides a method for diagnosing or detecting cancer by measuring the expression level of the SUV39H2 gene in a subject-derived sample. Examples of cancer diagnosed or detected by the method of the present invention include lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. In the context of the present invention, lung cancer includes NSCLC and SCLC. In addition, NSCLC includes lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) and lung large cell carcinoma (LCC).

  In the context of the present invention, the term “diagnosis” may include detection as well as prediction and likelihood analysis. A method for diagnosing or detecting cancer using the expression level of the SUV39H2 gene as an indicator of cancer in a subject-derived biological sample, wherein the SUV39H2 expression level is increased or increased in the sample compared to a control level It is one of the objects of the present invention to provide a method for indicating the presence or suspicion of cancer cells in a sample. The present invention is a method for detecting or identifying cancer cells in a subject-derived tissue sample, the method comprising the step of determining the expression level of the SUV39H2 gene in the tissue sample, wherein the comparison is with a normal control level. Thus, increased gene expression levels indicate the presence or suspicion of cancer cells in this tissue sample.

  According to the present invention, an intermediate result for examining the condition of an object, i.e. a method for monitoring an object may be provided. Such intermediate results can be combined with additional information to assist doctors, nurses, or other practitioners to diagnose that the subject suffers from the disease. Alternatively, the present invention may be used to detect cancerous cells in a subject-derived tissue, and can provide a doctor with useful information for diagnosing that the subject suffers from the disease. .

  Diagnostic methods may be used clinically in making therapeutic decisions, including therapeutic intervention, diagnostic criteria such as stage, and disease monitoring and monitoring of cancer. To improve diagnostic accuracy, the expression levels of other cancer-related genes, such as genes known to be differentially expressed in cancer, can also be measured. In addition, if the expression levels of multiple cancer-related genes are compared, the subject is affected by lung cancer because the gene expression pattern is similar between the sample and the cancerous reference, or Indicates that there is a risk of developing lung cancer.

  Thus, to assist a physician, nurse, or other medical practitioner in determining whether a test subject has the disease, the results of SUV39H2 expression may be supplemented with additional information or other diagnostic information. May be combined with an indicator, these include histopathology, levels of known tumor markers in the blood, and the clinical course of the subject. For example, according to the present invention, the expression level of the SUV39H2 gene can be used in combination with other diagnostic indicators including histopathology, levels of known tumor markers in the blood and clinical course of the subject, etc. . For example, some of the well-known diagnostic lung cancer markers in blood include CYFRA, ProGRP, NSC, SLX, CA19-9, CEA, TPA and BFP, and cervical cancer markers in blood include STN, CA72 -4 and SCC, bladder cancer markers in blood include NMP22, BFP, TPA, and esophageal cancer markers in blood include SCC, CEA, TPA, BFP and CA19-9, Examples of prostate cancer markers include PSA, PAP and BFP. Specifically, in this particular embodiment of the invention, the results of gene expression analysis serve as intermediate results for further diagnosis of the subject's disease state.

Particularly preferred embodiments of the present invention are described in the following items [1] to [11]:
[1] A method for detecting or diagnosing cancer in a subject, comprising the step of determining the expression level of the SUV39H2 gene in a subject-derived biological sample, wherein the gene is compared to a normal control level of the gene, The increased level is a method, or a method for detecting or diagnosing cancer in a subject, which indicates that the subject is suffering from or at risk of developing cancer, comprising the following steps: Method:
(I) isolating or recovering a subject-derived biological sample;
(Ii) contacting the subject-derived biological sample with an oligonucleotide that hybridizes to the SUV39H2 polynucleotide or an antibody that binds to the SUV39H2 polypeptide to measure or determine the expression level of the SUV39H2 gene; and (iii) ) Measuring or determining the expression level of the SUV39H2 gene based on this contact, wherein said increased level compared to the normal control level of the SUV39H2 gene is that the subject is suffering from cancer or A method to show that you have a risk of developing cancer;
[2] The method according to [1] above, wherein the measured sample expression level is at least 10% higher than the normal control level;
[3] The method according to [1] or [2] above, wherein the expression level is detected by a method selected from the following:
(A) a method for detecting mRNA of the SUV39H2 gene,
(B) a method for detecting a protein encoded by the SUV39H2 gene, and (c) a method for detecting a biological activity of the protein encoded by the SUV39H2 gene;
[4] The method according to [3] above, wherein the biological activity is cell growth promoting activity or methyltransferase activity;
[5] The method according to [3], wherein the expression level is determined by detecting mRNA with a probe for mRNA;
[6] The method according to [3] above, wherein the expression level is determined by detecting a protein with an antibody against the protein;
[7] The cancer according to any one of [1] to [6], wherein the cancer is selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor cancer. Described method;
[8] The method according to any one of [1] to [7], wherein the biological sample derived from the subject is a biopsy specimen, saliva, sputum, blood, serum, plasma, pleural effusion, or urine sample. ;
[9] The method according to any one of [1] to [8] above, wherein the subject-derived biological sample contains epithelial cells;
[10] The method according to any one of [1] to [9] above, wherein the subject-derived biological sample contains cancer cells; and [11] the subject-derived biological sample is The method according to any one of [1] to [10], wherein the method comprises cancerous epithelial cells.

Methods for diagnosing cancer are described in more detail below. The subject to be diagnosed according to the present invention is preferably a mammal. Exemplary mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.
The method of the invention preferably utilizes a biological sample obtained or collected from the subject to be diagnosed. Any biological material can be used as a biological sample for determination as long as it can contain the desired transcriptional or translational product of the SUV39H2 gene. Examples of suitable subject-derived biological samples include, but are not limited to, body tissues such as biopsy specimens and body fluids such as blood, sputum and urine. Preferably, the biological sample contains a cell population comprising epithelial cells, more preferably cancerous epithelial cells or epithelial cells from tissue suspected of being cancerous. Further, if necessary, the cells may be purified from the resulting body tissue and fluid and then used as a biological sample.

  In the context of the present invention, any cancer that overexpresses the SUV39H2 gene can be diagnosed or detected by the method of the present invention. Preferred cancers to be diagnosed or detected include lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. In order to diagnose or detect such cancers, subject-derived biological samples are preferably collected from the following organs: lung for lung cancer; cervix for cervical cancer; bladder for bladder cancer; esophagus Esophageal for cancer; bone for osteosarcoma; prostate for prostate cancer; and soft tissue for soft tumor.

  Preferably, the subject-derived biological sample is recovered from an area suspected of being cancerous in the organ. Thus, lung tissue samples, cervical tissue samples, bladder tissue samples, esophageal tissue, bone tissue, prostate tissue, or soft tissue samples collected from suspicious areas are preferably lung cancer, cervical cancer, bladder cancer, respectively, It can be used as a subject-derived biological sample for diagnosis or detection of esophageal cancer, osteosarcoma, prostate cancer or soft tissue tumor. Such tissue samples can be obtained by biopsy or surgical excision.

  According to the present invention, the expression level of the SUV39H2 gene in a subject-derived biological sample is determined by comparison to a control sample and then correlated with a particular health or disease state. The expression level of the SUV39H2 gene can be determined at the transcription product level using methods known in the art. For example, SUV39H2 mRNA may be quantified using a probe by a hybridization method (eg, Northern hybridization). Detection may be performed on a chip or array. Use of an array is preferable for detecting the expression level of a plurality of genes (for example, various cancer-specific genes) including the SUV39H2 gene. One skilled in the art can prepare such probes utilizing known sequence information for the SUV39H2 gene. For example, SUV39H2 gene cDNA may be used as a probe. If necessary, the probe may be labeled with a suitable label, such as a dye, fluorescence and radioisotope, and the expression level of the gene may be detected as the intensity of the hybridized label.

  Alternatively, the transcription product of the SUV39H2 gene may be quantified using primers by an amplification-based detection method (eg, RT-PCR). Such primers can also be prepared based on available sequence information of the SUV39H2 gene. For example, the primers (SEQ ID NOs: 5 and 6, for SUV39H2) used in the examples may be used for detection by RT-PCR or Northern blot, but the present invention is not limited thereto.

  Probes or primers suitable for use in the context of the method of the invention will hybridize to SUV39H2 mRNA under stringent, moderate stringent or low stringency conditions. Details of “stringent conditions” are described in the section entitled “Genes and Polypeptides”. Alternatively, diagnosis may involve detection of SUV39H2 translation products (ie, polypeptides or proteins) using methods known in the art. For example, the amount of SUV39H2 protein may be determined using an antibody against the SUV39H2 polypeptide and correlated with a disease or normal condition. Here, the “antibody against SUV39H2 polypeptide” refers to an antibody produced against the SUV39H2 polypeptide or a fragment thereof and specifically binding to the SUV39H2 polypeptide. Here, “specifically binds to SUV39H2 polypeptide” means that the antibody binds to SUV39H2 polypeptide but does not detectably bind to other polypeptides.

  The amount of translation product / protein may be determined, for example, using an immunoassay that uses an antibody that specifically recognizes the protein. An antibody suitable for use in the context of the method of the invention may be monoclonal or polyclonal. Furthermore, any immunological fragment or modified antibody of an antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) retains the ability of that fragment or modified antibody to bind to SUV39H2 protein. Insofar as it may be used for detection. Methods for preparing these types of antibodies for the detection of proteins are well known in the art and any method can be utilized in the present invention to prepare such antibodies and their equivalents. Also good.

  Alternatively, the expression level of the SUV39H2 gene may be determined based on its translation product, for example, through an examination of staining intensity observed through immunohistochemical analysis using antibodies against the SUV39H2 protein. More specifically, the observation of intense staining indicates an increased presence of protein and at the same time a high expression level of the SUV39H2 gene.

Further, the translation product may be determined based on its biological activity. As discovered herein, SUV39H2 protein has been shown to be involved in the growth of cancer cells. Therefore, the cancer cell growth promoting activity of SUV39H2 protein may be used as an indicator of SUV39H2 protein present in a biological sample. Alternatively, the methyltransferase activity of the SUV39H2 protein may also be used to determine the expression level of the SUV39H2 gene.
In addition to the expression level of the SUV39H2 gene, the expression levels of other cancer-related genes, such as genes that are known to be differentially expressed in cancer, are also determined to confirm the diagnosis. Also good.

  In the context of the present invention, a method for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begins with the determination of the expression level of the SUV39H2 gene. This expression level may be determined by any of the techniques described above. Once determined, this level can then be compared to a control level. In the context of the present invention, the gene expression level is, for example, 10%, 25%, or 50%, or at least 0.1 fold, at least 0.2 fold, at least 0. 0, compared to the control level. A change or increase of 5 times, at least 2 times, at least 5 times, or at least 10 times or more is recognized as “changed” or “increased”. Thus, if the expression level of a cancer marker gene comprising a SUV39H2 gene in a biological sample is increased by, for example, 10%, 25%, or 50% from the control level of the corresponding lung cancer marker gene, or An increase is considered to be greater than 1 fold, 1.5 fold, 2.0 fold, 5.0 fold, 10.0 fold, or even higher.

  In the context of the present invention, the phrase “control level” refers to the expression level of the SUV39H2 gene detected in a control sample and includes both normal and cancer control levels. The phrase “normal control level” refers to the level of SUV39H2 gene expression detected in a population of normal healthy individuals or those who are known not to have cancer. A healthy person is one who has no clinical symptoms of cancer. Normal control levels can be determined using normal cells obtained from non-cancerous tissue. The “normal control level” may also be the expression level of the SUV39H2 gene detected in a person who is known not to have cancer or in normal healthy tissue or cells of a population. On the other hand, the phrase “cancer control level” or “cancerous control level” refers to the expression level of the SUV39H2 gene detected in cancerous tissues or cells of a person or population suffering from cancer.

  An increase in the expression level of SUV39H2 detected in a subject-derived sample compared to a normal control level is an indication that the subject (from which the sample is obtained) is suffering from or at risk of developing cancer. It shows having. In the context of the present invention, a subject-derived sample can be any tissue obtained from a test subject, eg, a patient known or suspected of having cancer. For example, the tissue may include epithelial cells. More specifically, the tissue may be an epithelial cell suspected of being cancerous. Similarity between the expression level of the sample and the cancer control level indicates that the subject (from which the sample is obtained) is suffering from or at risk of developing cancer. When the expression levels of other cancer-related genes are also measured and compared, the similarity in the gene expression pattern between the sample and the cancerous reference may indicate that the subject has cancer or cancer Indicates that there is a risk of onset.

  The control level may be determined at the same time as the test biological sample, using a sample previously collected and stored from a subject whose disease state (cancerous or non-cancerous) is known. Alternatively, the control level is determined by a statistical method based on the results obtained by analyzing the expression level of the SUV39H2 gene in a sample from a subject whose disease state is known in advance. Also good. Furthermore, the control level may be a database of expression patterns from previously tested cells. Moreover, according to one aspect of the invention, the expression level of the SUV39H2 gene in a biological sample may be compared to a plurality of control levels, which are determined from a plurality of reference samples. It is preferred to use a control level determined from a reference sample from a tissue type similar to the subject-derived biological sample. Moreover, it is preferred to use a standard value for the expression level of the SUV39H2 gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, an average value ± 2SD or an average value ± 3SD range may be used as the standard value.

  If the expression level of the SUV39H2 gene is increased compared to the normal control level, or similar to the cancerous control level, the subject is suffering from or at risk of developing cancer Can be diagnosed. In addition, when comparing the expression levels of multiple cancer-related genes, the similarity in gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from cancer or has It shows that there is a risk of developing.

Housekeeping in which the difference between the expression level of the test biological sample and the control level is known not to differ depending on the control nucleic acid, eg, the cancerous or non-cancerous state of the cell It can be normalized to the expression level of the gene. Exemplary control genes include, but are not limited to, β-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
In order to facilitate the use described above, the present invention provides a diagnostic reagent for diagnosing cancer. Such a reagent can be selected from:
(A) a reagent for detecting mRNA of the SUV39H2 gene;
(B) a reagent for detecting SUV39H2 protein; and (c) a reagent for detecting biological activity of SUV39H2 protein.

  Specifically, such a reagent is an oligonucleotide that hybridizes to the transcription product of the SUV39H2 gene, or an antibody that binds to a SUV39H2 polypeptide. In other words, the present invention also provides a kit for use in the diagnosis or detection of cancer, wherein the kit comprises a reagent that binds to a transcription or translation product of the SUV39H2 gene.

The findings of the present invention reveal that SUV39H2 is not only a useful diagnostic marker but also a target for cancer treatment. Therefore, cancer treatment targeting SUV39H2 can be achieved by the present invention. In the context of the present invention, the phrase “cancer therapy targeting SUV39H2” refers to the suppression or inhibition of SUV39H2 activity and / or expression in cancer cells. Any anti-SUV39H2 agent can be used for cancer treatment targeting SUV39H2. In the context of the present invention, preferred anti-SUV39H2 agents comprise the following substances as active ingredients:
(A) the double-stranded molecule of the present invention,
(B) DNA encoding it, or (c) a vector encoding it. All of them are discussed in detail below.

Thus, in a preferred embodiment, the present invention provides a method of (i) diagnosing whether a subject has a cancer to be treated and / or (ii) selecting a subject for cancer treatment; These methods include the following steps:
(A) determining the expression level of SUV39H2 in a cancer cell or tissue obtained from a subject suspected of having a cancer to be treated;
(B) comparing the expression level of this SUV39H2 with a normal control level;
(C) diagnosing the subject as having a cancer to be treated if the SUV39H2 expression level is increased compared to a normal control level; and (d) in step (c), the subject If the patient is diagnosed with cancer to be treated, selecting the subject for cancer treatment.

Alternatively, such a method comprises the following steps:
(A) determining the expression level of SUV39H2 in a cancer cell or tissue obtained from a subject suspected of having a cancer to be treated;
(B) comparing the expression level of SUV39H2 with a cancerous control level;
(C) diagnosing the subject as having a cancer to be treated if the SUV39H2 expression level is similar or equivalent to a cancerous control level; and (d) in step (c), the subject If the patient is diagnosed with cancer to be treated, selecting the subject for cancer treatment.

Exemplary cancers include, but are not limited to, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and / or soft tissue tumor. Therefore, prior to administration of the double-stranded molecule of the invention as an active ingredient, it is ascertained whether the expression level of SUV39H2 in the cancer cell or tissue to be treated is enhanced compared to normal cells of the same organ It is preferable. Thus, in one aspect, the invention provides a method of treating a cancer that (over) expresses SUV39H2, which method may comprise the following steps:
i) determining the expression level of SUV39H2 in a cancer cell or tissue obtained from a subject having a cancer to be treated;
ii) comparing the expression level of this SUV39H2 with a normal control; and
iii) In subjects with cancers that overexpress SUV39H2 compared to normal controls,
(A) the double-stranded molecule of the present invention,
Administering at least one component selected from the group consisting of (b) DNA encoding it, and (c) a vector encoding it. Alternatively, the present invention also
(A) the double-stranded molecule of the present invention,
A medicament for use in administration to a subject having cancer that overexpresses SUV39H2, comprising (b) DNA encoding the same and (c) at least one component selected from the group consisting of vectors encoding the same A composition is provided. In other words, the present invention further provides:
(A) the double-stranded molecule of the present invention,
Provided is a method for identifying a subject to be treated with (b) DNA encoding it, or (c) a vector encoding it.

  Such a method may comprise the step of determining the expression level of SUV39H2 in a cancer cell or tissue from the subject, wherein the increase in the level compared to the normal control level of this gene Indicates that the subject has a cancer that may be treated with the inventive double-stranded molecule.

The cancer treatment method of the present invention is described in more detail below.
The subject to be treated by the method of the present invention is preferably a mammal. Exemplary mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.
According to the present invention, the expression level of SUV39H2 in a cancer cell or tissue obtained from a subject is determined using methods known in the art, for example, at the transcription (nucleic acid) product level. For example, SUV39H2 transcript levels can be determined using hybridization methods (eg, Northern hybridization), chips or arrays, probes, RT-PCR.

Alternatively, translation products may be detected for the treatment of the present invention. For example, the amount of protein observed (SEQ ID NOs: 22, 24, 26, 28, and 30) may be determined.
As another method of detecting the expression level of the SUV39H2 gene based on the translation product, the intensity of staining may be measured through immunohistochemical analysis using an antibody against the SUV39H2 protein. Specifically, in this measurement, intense staining indicates an increase in the presence / level of protein and at the same time a high expression level of the SUV39H2 gene.
Methods for the detection or measurement of SUV39H2 polypeptide and / or the polynucleotide encoding it can be exemplified as described above.

Kit for diagnosing cancer:
The present invention provides a kit for diagnosing or detecting cancer. Preferably, the cancer may be selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor.
The kit of the invention comprises at least one reagent for detecting the expression level of the SUV39H2 gene in a subject-derived biological sample, which reagent may be selected from the group consisting of:
(A) a reagent for detecting mRNA of the SUV39H2 gene;
(B) a reagent for detecting SUV39H2 protein; and (c) a reagent for detecting biological activity of SUV39H2 protein.

  Suitable reagents for detecting SUV39H2 mRNA include nucleic acids that specifically bind to or identify SUV39H2 mRNA, such as oligonucleotides having sequences complementary to a portion of SUV39H2 mRNA. These types of oligonucleotides are exemplified by primers and probes that are specific for SUV39H2 mRNA. These types of oligonucleotides may be prepared based on methods well known in the art. If necessary, the reagent for detecting SUV39H2 mRNA may be immobilized on a solid substrate. Moreover, two or more reagents for detecting SUV39H2 mRNA may be included in the kit.

  The probe or primer of the present invention typically comprises a substantially purified oligonucleotide. The oligonucleotide is typically at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, under stringent conditions. Or 25 consecutive regions of a nucleotide sequence that hybridizes to a sense strand nucleotide sequence of a nucleic acid comprising an SUV39H2 sequence, or an antisense strand nucleotide sequence of a nucleic acid comprising an SUV39H2 sequence, or a natural mutant of these sequences. Including. In particular, for example, in a preferred embodiment, an oligonucleotide having a length of 5 to 50 can be used as a primer for amplifying the gene to be detected. More preferably, SUV39H2 gene mRNA or cDNA can be detected using an oligonucleotide probe or primer having a length of 15-30 bp. In a preferred embodiment, the length of the oligonucleotide probe or primer can be selected from 15-25 bp. Assay procedures, devices, or reagents for the detection of genes using such oligonucleotide probes or primers are well known (eg, oligonucleotide microarray or PCR). In these assays, the probe or primer can also include a tag or linker sequence. In addition, the probe or primer can be modified with a detectable label or an affinity ligand to be captured. Alternatively, in a hybridization-based detection procedure, a polynucleotide having a length of several hundred (eg, about 100-200) bases to several kilos (eg, about 1000-2000) bases is converted into a probe (eg, Northern It can also be used for blotting assays or cDNA microarray analysis).

  On the other hand, a suitable reagent for detecting SUV39H2 protein includes an antibody against SUV39H2 protein. The antibody may be monoclonal or polyclonal. In addition, any immunological fragment or modified antibody of an antibody (eg, chimeric antibody, scFv, Fab, F (ab ′) 2, Fv, etc.) retains the ability of that fragment or modified antibody to bind to SUV39H2 protein. As long as it does, you may use as a reagent. Methods for preparing such antibodies for the detection of proteins are well known in the art and any method can be used in the present invention to prepare such antibodies and their equivalents. Good.

  The antibody may be labeled with a signal generating molecule via direct linkage or indirect labeling techniques. Labels and methods for labeling antibodies and detecting binding of antibodies to their targets are well known in the art, and any label and method may be used for the present invention. In addition, two or more reagents for detecting SUV39H2 protein may be included in the kit.

  Alternatively, the expression of SUV39H2 protein in a biological sample may be detected or measured using its biological activity as an indicator. Biological activity can be determined, for example, by measuring cell proliferation activity by the expressed SUV39H2 protein in a biological sample. For example, the cell proliferation activity of a subject-derived biological sample can be determined by culturing cells in the presence of a subject-derived biological sample, detecting the rate of growth, measuring the cell cycle, or colonizing ability. It can be determined by measuring. More than one reagent for detecting the biological activity of SUV39H2 protein may be included in the kit.

  The kit may contain two or more of the reagents described above. In addition, the kit detects solid substrates and reagents for binding probes to SUV39H2 mRNA or antibodies to SUV39H2 protein, media and containers for culturing cells, positive and negative control reagents, and antibodies to SUV39H2 protein. A secondary antibody may be included. For example, a tissue sample obtained from a subject with or without cancer serves as a useful control reagent.

  The kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts including instructions for use ( For example, written documents, tapes, CD-ROMs, etc.). These reagents and others may be packaged in a labeled container. Suitable containers include bottles, vials and test tubes. The container may be formed from a variety of materials such as glass or plastic.

  As one aspect of the present invention, when the reagent is a probe for SUV39H2 mRNA, the reagent may be immobilized on a solid substrate such as a porous strip to form at least one detection site. The measurement or detection region of the porous strip can contain multiple sites, each containing a nucleic acid (probe). The test strip may also include sites for negative and / or positive controls. Alternatively, the control site may be located on a strip that is separated from the test strip. In addition, different detection sites may contain different amounts of immobilized nucleic acid, ie, higher amounts in the first detection site and sequentially lower amounts in subsequent sites. Upon addition of the test sample, the number of sites exhibiting a detectable signal provides a quantitative indication of the amount of SUV39H2 mRNA present in the sample. The detection site may be designed in any suitable detectable shape and is typically a rod or dot shape across the width of the test strip.

  The kit of the present invention may further contain a positive control sample. The positive control samples of the present invention may be prepared by collecting SUV39H2 positive samples and then assaying these SUV39H2 levels. In one embodiment, the SUV39H2-positive tissue sample may be composed of cancer cells that express SUV39H2. Such cancer cells include, but are not limited to, cancer selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. The SUV39H2 level of the positive control sample can be, for example, above the cutoff value.

  Alternatively, the SUV39H2-positive sample may be a clinical cancer tissue obtained from a cancer patient. Such cancers include, but are not limited to, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. Alternatively, a positive control sample may be prepared by determining a cutoff value and preparing a sample containing an amount of SUV39H2 mRNA or protein above this cutoff value. Here, the phrase “cut-off value” refers to a value that divides between the normal range and the cancerous range. For example, those skilled in the art may determine the cutoff value using a receiver operating characteristic (ROC) curve.

  The kit of the present invention may additionally or alternatively include a SUV39H2 standard sample that provides an amount of cut-off value of SUV39H2 mRNA or polypeptide. The kit of the invention may further comprise a negative control sample, such as a blood sample from a non-SUV39H2-expressing cell or tissue, or a control without cancer. In the context of the present invention, a negative control sample is a non-cancerous cell line or non-cancerous such as normal lung tissue, cervical tissue, bladder tissue, esophageal tissue, bone tissue, prostate tissue and soft tissue. It may be prepared from tissue or by preparing a sample containing SUV39H2 mRNA or protein below the cutoff value.

Anti-cancer screening:
The SUV39H2 gene, SUV39H2 polypeptide or functional equivalent thereof, or the transcriptional regulatory region of the gene can be used to screen for substances that alter the expression of the SUV39H2 gene or the biological activity of the SUV39H2 polypeptide. . Such a substance may be used as a drug candidate for treating or preventing a disease associated with SUV39H2 overexpression such as cancer. Accordingly, the present invention provides a method for screening candidate substances for the treatment or prevention of cancer using the SUV39H2 gene, SUV39H2 polypeptide or functional equivalent thereof, or the transcriptional regulatory region of the SUV39H2 gene. To do.

  Substances isolated and identified by the screening methods of the present invention as suitable candidates are expected to reduce, suppress and / or inhibit the expression of SUV39H2 gene or the activity of translation products of SUV39H2 gene, and thus cancer Candidates for the treatment and / or prevention of (especially lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor). Specifically, a substance screened through the methods of the present invention may be identified as having clinical efficacy and further tested for its ability to limit or prevent cancer cell growth in an animal model or test subject. it can.

  In the context of the present invention, the substance to be identified through the screening method of the invention may be any substance or a composition comprising several substances. Furthermore, the test substance exposed to the cell or protein according to the screening method of the present invention may be a single substance or a combination of substances. When combinations of materials are used in these methods, the materials may be contacted sequentially or simultaneously.

  Any test substance such as cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptide substance, synthetic micromolecular substance (anti-antigen) (Including nucleic acid constructs such as sense RNA, siRNA, ribozyme, and aptamer) and natural substances can be used in the screening method of the present invention. Test substances of the present invention can also be obtained using any of a number of combinatorial library methods known in the art, including (1) biological libraries, (2 ) Spatially addressable parallel solid phase library or liquid phase library, (3) synthetic library method requiring deconvolution, (4) “one bead one substance” library method and (5) affinity chromatography. Synthetic library methods using selection. Biological library methods using affinity chromatography selection are limited to peptide libraries, but the other four approaches can be applied to small libraries of peptides, non-peptide oligomers or compounds (Lam, Anticancer Drug Des 1997, 12: 145-67)). Examples of methods for the synthesis of molecular libraries can be found in the prior art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Compound libraries can be in solution (see Houghten, Bio / Techniques 1992, 13: 412-21), or beads (Lam, Nature 1991, 354: 82-4, chips (Fodor. Nature 1993, 364: 555-6). ), Bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Patent Application No. 2002103360).

A compound obtained by converting a part of the structure of the substance screened by the screening method of the present invention by addition, deletion and / or substitution is included in the substance obtained by the screening method of the present invention.
Furthermore, if the test substance screened is a protein, the entire amino acid sequence of the protein may be determined in order to estimate the nucleic acid sequence encoding the protein in order to obtain DNA encoding the protein. Or analyzing a partial amino acid sequence of the obtained protein to prepare an oligo DNA as a probe based on the sequence, and screening a cDNA library to obtain a DNA encoding the protein using the probe. Also good. The obtained DNA is confirmed for its usefulness in preparing test substances that are candidates for treating or preventing cancer or its postoperative recurrence or for inhibiting cancer cell growth. Test substances useful in the screening described herein include antibodies that specifically bind to SUV39H2 protein, or partial peptides thereof that lack the biological activity of the original protein in vivo.

Although the construction of a test substance library is well known in the art, the following provides further guidance in such substance library construction and test substance identification for the screening methods of the present invention.
It is now demonstrated that suppression of either the expression level or biological activity of SUV39H2 protein results in suppression of cancer cell growth. Thus, if a substance suppresses SUV39H2 expression and / or activity, such suppression indicates a potential therapeutic effect in the subject. In the context of the present invention, a potential therapeutic effect refers to clinical efficacy with reasonable expectations. Examples of such clinical efficacy include, but are not limited to:
(A) Reducing the expression of the SUV39H2 gene,
(B) a reduction in the size, prevalence, or metastatic potential of cancer in the subject,
(C) prevention of cancer formation, or (d) prevention or alleviation of clinical symptoms of cancer.

(I) Molecular modeling:
Construction of a test substance library is facilitated by knowledge of the molecular structure of a compound known to have the desired property and / or the molecular structure of SUV39H2. One approach for preliminary screening of test substances suitable for further evaluation is computer modeling of the interaction between the test substance and its target.

  Computer modeling techniques allow the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. This three-dimensional construct typically relies on X-ray crystallographic or NMR imaging data of the selected molecule. Molecular dynamics requires force field data. Computer graphic systems allow prediction of how new compounds are linked to target molecules and allow experimental manipulation of compounds and target molecule structures to full binding specificity. Predicting what a molecule-compound interaction will look like if one or both small changes are made typically has an easy-to-use menu-style interface between the molecular design program and the user Molecular mechanics software and computationally intensive computers are required.

  Examples of molecular modeling systems generally described above include the CHARMM and QUANTA programs (Polygen Corporation, Waltham, Mass). CHARMM performs energy minimization and molecular dynamics functions. QUANTA performs molecular structure building, graphic modeling and analysis. QUANTA allows interactive construction, modification, visualization, and analysis of intermolecular behavior.

  A number of articles discuss computer modeling of drugs that interact with specific proteins, for example, Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; And Askew et al., J Am Chem Soc 1989, 111: 1082-90 for model receptors for nucleic acid components.

  Other computer programs to screen and diagram chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario It is. For example, DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17: 266-78; Shoichet et al., Science 1993, 259: 1445-50.

  Once the putative inhibitor is identified, any number of variants can be constructed based on the chemical structure of the identified putative inhibitor using combinatorial chemistry techniques, as described below. The resulting library of putative inhibitors, or “test substances”, is a test to treat or prevent cancers such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. To identify substances, they can be screened using the methods of the present invention.

(Ii) Combinatorial chemical synthesis:
A combinatorial library of test substances may be created as part of a rational drug design program that includes knowledge of the core structure present in known inhibitors. This approach allows the library to be maintained at a reasonable size and facilitates high throughput screening. Alternatively, a simple, particularly short polymer molecule library may be constructed by simply synthesizing all permutations of the molecular family that make up the library. An example of this latter approach is a library of all peptides 6 amino acids long. Such a peptide library can contain a permutation of all 6 amino acid sequences. This type of library is called a linear combinatorial chemical library.

The preparation of combinatorial chemical libraries is well known to those skilled in the art and may be made by either chemical synthesis or biological synthesis. Combinatorial chemical libraries include peptide libraries (eg, US Pat. No. 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354:
84-6), but is not limited to these. Other chemistries for generating chemical diversity libraries can also be used. Such chemistry includes, but is not limited to: peptides (eg, International Publication No. WO 91/19735), encoded peptides (eg, WO 93/20242), random biooligomers (eg, WO92 / 00091), benzodiazepines (eg, US Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909). -13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), non-peptidic peptidomimetics with a glucose skeleton (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), similar organic synthesis of low compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), Rigocarbamate (Cho et al., Science 1993, 261: 1303) and / or peptidyl phosphonate (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid library (Ausubel, Current Protocols in Molecular Biology 1995 supplement) Sambrook et al., Molecular Cloning: See A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083) ), Antibody libraries (see, eg, Vaughan et al., Nature Biotechnology 1996, 14 (3): 309-14 and PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al., Science 1996, 274: 1520-22; see US Pat. No. 5,593,853), and small organic libraries (eg, benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1; 6 (6): 624-31 .; isoprenoids, US Pat. No. 5,569,588; thiazolidinones and metathiazanones, US Pat. No. 5,549,974; Pyrrolidines, see US Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, US Pat. No. 5,506,337; benzodiazepines, 5,288,514, etc.).

  Materials and methods for the preparation of combinatorial libraries are commercially available (eg, 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA , 9050 Plus, Millipore, Bedford, MA). In addition, a number of combinatorial libraries are commercially available per se. (See, eg, ComGenex, Princeton N.J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

(Iii)
Other candidates:
Another approach uses a recombinant bacteriophage to create a library. "Phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6) Can be used to build very large libraries (eg, 10 ⁶ to 10 ⁸ chemicals). The second approach primarily uses chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); And the method of Fodor et al. (Science 1991, 251: 767-73) is exemplary. Furka et al. (14th International Congress of Biochemistry 1988, Volume # 5, Abstract FR: 013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Pat. No. 4,631, 211) and Rutter et al. (US Pat. No. 5,010,175) describes a method for producing a mixture of peptides that can be tested as agonists or antagonists.

Aptamers are macromolecules composed of nucleic acids that bind tightly to specific molecular targets. Tuerk and Gold (Science. 249: 505-510 (1990)) disclose a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for aptamer selection. In the SELEX method, a large library of nucleic acid molecules (eg, 10 ¹⁵ different molecules) can be used for screening.

In addition to the full-length SUV39H2 polypeptide, fragments of the polypeptide may be used for the screening methods of the invention as long as the fragment used retains at least one biological activity of the native SUV39H2 polypeptide. May be. Examples of such biological activities contemplated by the present invention include cell growth promoting and methyltransferase activities of native SUV39H2 polypeptides.
The SUV39H2 polypeptide or functional equivalent thereof may be further linked to other substances as long as the resulting polypeptide retains at least one biological activity of the native SUV39H2 polypeptide. Useful substances include peptides, lipids, saccharides and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These types of modifications may be made to confer additional functions or to stabilize polypeptides and fragments.

(I) General screening method:
The transcriptional regulatory region of SUV39H2 gene, SUV39H2 polypeptide or SUV39H2 gene can be used to identify substances that alter SUV39H2 gene expression or biological activity of SUV39H2 polypeptide. As revealed herein, the SUV39H2 gene is overexpressed in cancer and is involved in the growth and / or survival of cancer cells. Therefore, a substance that alters the expression of the SUV39H2 gene or the biological activity of the SUV39H2 polypeptide can be a candidate for a therapeutic agent for cancer.

  An antagonist that binds to a SUV39H2 polypeptide can reduce, suppress or inhibit a biological activity that mediates cancer cell growth. As such, they are potential candidates for treating cancer. Therefore, the present invention provides a method for identifying a candidate for treating or preventing cancer expressing SUV39H2 gene by identifying a substance that binds to SUV39H2 polypeptide.

In some embodiments, the SUV39H2 polypeptide may be immobilized on a suitable support. Examples of supports that can be used for protein binding include, for example, insoluble polysaccharides such as agarose, cellulose and dextran; and synthetic resins such as polyacrylamide, polystyrene and silicon; Commercially available beads and plates (eg, multiwell plates, biosensor chips, etc.) can be used. If beads are used, they can be packed into a column. Alternatively, the use of magnetic beads is also known in the art, making it possible to easily isolate proteins bound on the beads via magnetic force.
Binding of the protein to the support can be done according to conventional methods such as, for example, chemical binding and physical adsorption. Alternatively, the protein can be bound to the support via an antibody that specifically recognizes the protein. Moreover, protein binding to the support may be performed using avidin and biotin.

  Immobilized polypeptides can be exposed to synthetic compounds, natural material banks, or random phage peptide display libraries, not only proteins but also compounds that bind to the protein (including agonists and antagonists). High-throughput screening methods based on combinatorial libraries for release (Wrighton et al., Science 273: 458-63 (1996); Verdine, Nature 384: 11-3 (1996)) are well known to those skilled in the art. is there.

Furthermore, in the screening method of the present invention, a substance that suppresses the expression level of the SUV39H2 gene can also be identified as a cancer treatment candidate. The expression level of a polypeptide or functional equivalent thereof can be detected according to any method known in the art. For example, a reporter assay can be used. Suitable reporter genes and host cells are well known in the art. The reporter construct required for screening can be prepared using the transcriptional regulatory region of the SUV39H2 gene. When the transcriptional regulatory region of a gene is known to those skilled in the art, reporter constructs can be prepared using conventional sequence information. If a transcriptional regulatory region has not been identified, a nucleotide segment comprising the transcriptional regulatory region can be isolated from a genomic library based on the nucleotide sequence information of that gene. Specifically, the reporter construct required for screening can be prepared by connecting a reporter gene sequence to the transcriptional regulatory region of the SUV39H2 gene. The transcriptional regulatory region of the SUV39H2 gene is a region at least 500 bp upstream, such as 1000 bp, such as 5000 or 10000 bp upstream from the start codon. Nucleotide segments containing transcriptional regulatory regions can be isolated from genomic libraries or can be amplified by PCR. Methods and assay protocols for identifying transcriptional regulatory regions are also well known (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., Chapter 17, 2001, Cold Springs Harbor Laboratory Press).
Furthermore, in the screening method of the present invention, a substance that inhibits the biological activity of SUV39H2 polypeptide can also be identified as a candidate for a therapeutic agent for cancer.

  A variety of low-throughput and high-throughput enzyme assay formats are known in the art and can be readily adapted for detection or measurement of biological activity of SUV39H2 polypeptides. For high throughput assays, the substrate is conveniently immobilized on a solid support. After the reaction, the substrate converted by the polypeptide can be detected on the solid support by the method described above. Alternatively, the contacting step can be performed in solution, after which the substrate can be immobilized on a solid support and the substrate converted by the polypeptide detected. To facilitate such assays, the solid support can be coated with streptavidin and the substrate can be labeled with biotin, or the solid support can be coated with an antibody against the substrate. One skilled in the art can determine a suitable assay format depending on the desired throughput capability of the screening.

  The assays of the present invention are also suitable for automated procedures that facilitate high throughput screening. A number of well-known robotic systems have been developed for solution phase chemistry. These systems include automated workstations such as automated synthesizers developed by Takeda Chemical Industries, Ltd. (Osaka, Japan), and numerous robotic systems that utilize robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass .; Orca, Hewlett Packard, Palo Alto, Calif.), Which mimics manual synthesis operations by chemists. Any of the above devices are suitable for use in the present invention. The nature and enforcement (if any) of modifications to these that will enable these devices to operate as discussed herein will be apparent to those skilled in the art. In addition, numerous combinatorial libraries are commercially available per se (eg, ComGenex, Princeton, NJ, Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D See Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

In the present invention, it becomes clear that suppression of the expression level of SUV39H2 gene or biological activity of SUV39H2 polypeptide results in suppression of cancer cell growth. Thus, if a substance suppresses SUV39H2 gene expression or SUV39H2 polypeptide activity, this suppression is an indication of a potential therapeutic effect in the subject. In the present invention, a potential therapeutic effect refers to clinical efficacy with reasonable expectations. In the present invention, such clinical efficacy includes the following:
(A) Reducing the expression of the SUV39H2 gene,
(B) a reduction in the size, morbidity, or metastatic potential of cancer in the subject;
(C) prevention of cancer formation, or (d) prevention or alleviation of clinical symptoms of cancer.

(II) Screening for substances that bind to SUV39H2 polypeptide:
In the course of the present invention, the SUV39H2 gene is overexpressed in various cancers such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor, but nevertheless excludes the testis It has been elucidated that there is little or no expression in normal organs (FIGS. 1, 2, 3, 6, 9, 10 and Tables 3, 4, 5, 6). Therefore, using SUV39H2 polypeptide,
The present invention provides a method of screening for a substance that binds to SUV39H2 polypeptide. Due to the expression of the SUV39H2 gene in cancer, substances that bind to the SUV39H2 polypeptide are expected to suppress the growth of cancer cells and are therefore useful for one or both of cancer treatment and prevention. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the growth of cancer cells, and a method for screening a candidate substance for one or both of cancer treatment and prevention using SUV39H2 polypeptide. Specifically, in one embodiment, the screening method of the present invention includes the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting a binding activity between the polypeptide or a functional equivalent thereof and the test substance; and (c) selecting a test substance that binds to the polypeptide or a functional equivalent.

According to the present invention, the therapeutic effect of a test substance on the treatment and prevention of cancer associated with inhibition of cancer cell proliferation and overexpression of SUV39H2 can be evaluated or estimated. Accordingly, the present invention also provides a method for screening candidate substances for inhibition of cancer cell growth or one or both of cancer treatment and prevention, which comprises the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting binding between the polypeptide (or functional equivalent) and the test substance; and (c) associating the binding of (b) with a potential therapeutic effect of the test substance.

  In the context of the present invention, the therapeutic effect may be related to the binding properties of the test substance. For example, if a test substance binds to a polypeptide (or functional equivalent), the test substance can be identified or selected as a candidate substance having a therapeutic effect. Alternatively, if the test substance does not bind to the polypeptide (or functional equivalent), the test substance can be identified as a substance that has no significant therapeutic effect.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance on one or both of cancer treatment and prevention associated with overexpression of SUV39H2 can be assessed or inferred as follows:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting the binding activity between the polypeptide or functional equivalent and the test substance; and (c) associating the potential therapeutic effect with the test substance, wherein the potential therapeutic effect is determined by whether the substance is a polypeptide or Shown when binding to functional equivalent.

  The method of the present invention is described in more detail below. The SUV39H2 polypeptide used in the screening method of the present invention may be a recombinant polypeptide, a naturally derived protein, or a partial peptide thereof. The polypeptide to be contacted with the test substance can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused to another polypeptide. In preferred embodiments, the polypeptide is isolated from SUV39H2-expressing cells or chemically synthesized for contact with a test substance in vitro.

  A number of methods well known to those skilled in the art can be used as a method of screening for proteins that bind to SUV39H2 polypeptide. Such screening can be performed, for example, by immunoprecipitation, specifically as follows. A gene encoding the SUV39H2 polypeptide is expressed in a host (eg, animal) cell or the like by inserting the gene into an expression vector for a foreign gene, such as pSV2neo, pcDNAI, pcDNA3.1, pCAGGS and pCD8.

  The promoter to be used for expression may be any promoter that can be used normally, such as the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141. (1982)), EF-α promoter (Kim et al., Gene 91: 217-23 (1990)), CAG promoter (Niwa et al., Gene 108: 193 (1991)), RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)), SR α promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), CMV early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365− 9 (1987)), SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), adenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946 (1989)), HSV TK Promoter, and the like.

  Introduction of a gene into a host cell for expressing a foreign gene can be performed according to any method, for example, electroporation (Chu et al., Nucleic Acids Res 15: 1311-26 (1987) ), Calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B., Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al Science 259: 230-4 (1993)).

  An SUV39H2 polypeptide can be expressed as a fusion protein containing a monoclonal antibody recognition site (epitope) by introducing an epitope of a monoclonal antibody whose specificity has been elucidated into the N-terminus or C-terminus of the polypeptide. it can. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors that can express a fusion protein with, for example, β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like by using a plurality of cloning sites are commercially available. Also provided herein are fusion proteins prepared by introducing only small epitopes composed of several to a dozen amino acids so that the fusion does not alter the properties of the SUV39H2 polypeptide. Polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV) -Tags), epitopes such as E-tag (epitope on monoclonal phage), and monoclonal antibodies that recognize them can be used as epitope-antibody systems for screening proteins that bind to SUV39H2 polypeptides. Yes (Experimental Medicine 13: 85-90 (1995)).

In the context of immunoprecipitation, immune complexes are formed by adding these antibodies to cell lysates prepared with an appropriate detergent. The immune complex is composed of a SUV39H2 polypeptide, a polypeptide capable of binding to the SUV39H2 polypeptide, and an antibody. In addition to using antibodies against the above epitopes, immunoprecipitation can also be performed using antibodies against SUV39H2 polypeptides, which can be prepared as described above. The immune complex can be precipitated by, for example, protein A sepharose or protein G sepharose when the antibody is a mouse IgG antibody. If the SUV39H2 polypeptide is prepared as a fusion protein with an epitope such as GST, the immunoconjugate can be prepared in the same manner as the use of antibodies against the SUV39H2 polypeptide in the same manner as glutathione-sepharose 4B. It can be formed using a substance that specifically binds to an epitope.
Immunoprecipitation can be performed, for example, according to literature methods (Harlow and Lane, Antibody, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteins, and bound proteins can be analyzed by protein molecular weight using an appropriate concentration of gel. Since proteins bound to SUV39H2 polypeptide are difficult to detect by common staining methods such as Coomassie staining or silver staining, the sensitivity of detection of this protein is that of the radioisotope, ³⁵ S-methionine or ³⁵ Improvement can be achieved by culturing the cells in a medium containing S-cysteine, labeling the protein in the cell, and detecting the protein. The target protein can be purified directly from an SDS-polyacrylamide gel and its sequence determined if the molecular weight of the protein is known.

  Alternatively, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used to screen for proteins that bind to SUV39H2 polypeptides. Specifically, the protein that binds to the SUV39H2 polypeptide is prepared using a phage vector (eg, ZAP) from a cultured cell that is expected to express the protein that binds to the SUV39H2 polypeptide. Express on LB agarose, immobilize the expressed protein on the filter, react the purified and labeled SUV39H2 polypeptide with the above filter and detect plaques expressing the protein bound to SUV39H2 polypeptide according to the label Can be obtained. An SUV39H2 polypeptide utilizes biotin and avidin binding, or utilizes an antibody that specifically binds to SUV39H2 polypeptide or a peptide or polypeptide fused to SUV39H2 polypeptide (eg, GST) It may be labeled. A method using a radioisotope or fluorescence may be used.

The terms “label” and “detectable label” are used herein to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. used. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (eg, DYNABEADS®), fluorescent dyes (eg, fluorescein, Texas red, rhodamine, green fluorescent protein, Etc.), radiolabels (eg ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P), enzymes (eg those commonly used in horseradish peroxidase, alkaline phosphatase and ELISA), and calorimetry (Calorimetric) labels such as colloidal gold or colored glass or plastic (eg polystyrene, polypropylene, latex, etc.) beads. Patents that teach the use of such labels include US Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 275, 149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters and fluorescent markers can be detected using a photodetector to detect luminescence. Enzymatic labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and the calorimetric label simply visualizes the colored label Is detected.

  Alternatively, in another embodiment, the screening method of the present invention comprises a two-hybrid cell system (“MATCHMAKER two-hybrid system”, “Mammalian MATCHMAKER two-hybrid assay kit”, “MATCHMAKER one-hybrid system” (Clontech); “HybriZAP two-hybrid” Vector system "(Stratagene); references" Dalton and Treisman, Cell 68: 597-612 (1992) "," Fields and Sternglanz, Trends Genet 10: 286-92 (1994) ").

  In the two-hybrid system, the SUV39H2 polypeptide is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein that binds to a SUV39H2 polypeptide so that when expressed, it is fused to a VP16 or GAL4 transcriptional activation region. Next, the cDNA library is introduced into the above yeast cell, and the cDNA derived from the library is isolated from the detected positive clone (when the protein that binds to the polypeptide of the present invention is expressed in the yeast cell). , The two bindings activate the reporter gene, making positive clones detectable). The protein encoded by this cDNA can be prepared by introducing the cDNA isolated above into E. coli and expressing the protein. Examples of suitable reporter genes include the Ade2 gene, lacZ gene, CAT gene, luciferase gene, and the like, but are not limited thereto, and these can be used in addition to the HIS3 gene.

  Substances that bind to the SUV39H2 polypeptide can also be screened using affinity chromatography. For example, the SUV39H2 polypeptide may be immobilized on a carrier on an affinity column, and a test substance containing a protein capable of binding to the polypeptide of the present invention is used for this column. Here, the test substance is, for example, a cell extract or a cell lysate. After loading the test substance, the column can be washed to prepare the substance bound to the polypeptide of the present invention. When the test substance is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, a cDNA library is screened using the oligo DNA as a probe, and the protein is encoded. DNA is obtained.

  A biosensor using the surface plasmon resonance phenomenon may be used as a means for detecting or quantifying the bound substance in the present invention. When using such a biosensor, the interaction between the SUV39H2 polypeptide and the test substance can be observed in real time as a surface plasmon resonance signal using only a small amount of the polypeptide and without labeling. Yes (for example, BIAcore, Pharmacia). Thus, a biosensor such as BIAcore can be used to assess the binding between the polypeptide of the invention and the test substance.

  Immobilized SUV39H2 polypeptides are exposed to synthetic compounds or natural substance banks or random phage peptide display libraries to isolate compounds that bind not only proteins but also SUV39H2 proteins (including agonists and antagonists). And screening methods that use high throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996) Hogan, Nature 384: 17-9 (1996)) is well known to those skilled in the art.

  In the present invention, it is elucidated that suppressing the expression level of the SUV39H2 gene reduces cell proliferation. Therefore, a candidate substance having a possibility of treating or preventing cancer can be identified by screening a candidate substance that binds to the SUV39H2 polypeptide. The possibility of treating or preventing cancer of these candidate substances may be assessed by a second and / or further screening to identify cancer therapeutic agents. For example, if a substance that binds to a SUV39H2 polypeptide inhibits the activity of a cancer, it may be concluded that such substance has a SUV39H2-specific therapeutic effect.

(III) Screening for substances that inhibit the biological activity of SUV39H2 polypeptide:
In the course of the present invention, the SUV39H2 gene has been shown to be highly overexpressed in various cancers including lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. .
Furthermore, suppression of the SUV39H2 gene by small interfering RNA (siRNA) resulted in cancer cell growth inhibition and / or cell death. Thus, it is clear that SUV39H2 polypeptides are involved in cancer cell survival, and therefore substances that inhibit the biological activity of SUV39H2 polypeptides are suitable candidate substances for cancer treatment.
Therefore, the present invention also provides a method for screening candidate substances for one or both of the treatment and prevention of SUV39H2-related cancer using the biological activity of SUV39H2 polypeptide as an indicator.
Exemplary SUV39H2-related cancers include lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor.

Specifically, the present invention provides the following methods:
A method for screening a candidate substance for one or both of cancer treatment and prevention, comprising the following steps:
(A) contacting the test substance with a SUV39H2 polynucleotide or a functional equivalent thereof;
(B) detecting the biological activity of the SUV39H2 polypeptide of step (a) or a functional equivalent thereof;
(C) comparing the biological activity detected in step (b) with that detected in the absence of the test substance;
(D) selecting a test substance that reduces or inhibits the biological activity of the SUV39H2 polypeptide or functional equivalent thereof.

In the context of the present invention for the therapeutic effect of a test substance on inhibiting the biological activity (eg cell growth promoting activity or methyltransferase activity) of SUV39H2 polypeptide and / or for the treatment or prevention of cancer Candidate substances may be evaluated. Accordingly, the present invention also provides a substance for inhibiting the biological activity of SUV39H2 polypeptide, or one or both of the treatment and prevention of SUV39H2-related cancer, using SUV39H2 polypeptide or a functional equivalent thereof. A method for screening a candidate substance, comprising the following steps:
(A) contacting a test substance with a SUV39H2 polypeptide or functional equivalent thereof; and (b) detecting the biological activity of the polypeptide or functional equivalent of step (a); and (c) ) (B) correlating the biological activity with the therapeutic effect of the test substance.

  In the context of the present invention, the therapeutic effect may be related to the biological activity of the SUV39H2 polypeptide or functional equivalent thereof. For example, if a test substance suppresses or inhibits the biological activity of a SUV39H2 polypeptide or functional equivalent thereof as compared to the level detected in the absence of the test substance, the test substance has a therapeutic effect. Can be identified or selected as a candidate substance. Alternatively, if the test substance does not suppress or inhibit the biological activity of the SUV39H2 polypeptide or functional equivalent thereof compared to the level detected in the absence of the test substance, the test substance is significant. It can be identified as a substance having no therapeutic effect.

Alternatively, in some embodiments, the present invention provides a method for assessing or estimating the therapeutic effect of a test substance on one or both of cancer treatment and prevention, or inhibition of cancer associated with SUV39H2 gene overexpression. And the method includes the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting the biological activity of the polypeptide of step (a) or a functional equivalent thereof; and (c) associating a potential therapeutic effect with the test substance, wherein the potential A therapeutic effect is when the substance inhibits the biological activity of the SUV39H2 polypeptide or a functional equivalent thereof compared to the biological activity of the polypeptide detected in the absence of the test substance, Indicated. In the context of expression, binding and biological activity, the term “suppressing” is used interchangeably with the terms “reducing” and “inhibiting” and provides partial to complete effects. including. Thus, as defined herein, the phrase “suppress biological activity” means at least 10% inhibition, more preferably at least 25%, inhibition of biological activity of SUV39H2 compared to the absence of the substance, Refers to 50% or 75% inhibition, and most preferably 90% inhibition. As described above, examples of such SUV39H2-related cancers include lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor.

  In the context of the present invention, the therapeutic effect may be related to the biological activity of the SUV39H2 polypeptide or functional equivalent thereof. For example, if a test substance suppresses or inhibits the biological activity of a SUV39H2 polypeptide or functional equivalent thereof as compared to the level detected in the absence of the test substance, the test substance has a therapeutic effect. May be identified or selected as a candidate substance. Alternatively, if the test substance does not suppress or inhibit the biological activity of the SUV39H2 polypeptide or functional equivalent thereof compared to the level detected in the absence of the test substance, the test substance is significant. It may be identified as a substance having no therapeutic effect.

  The method of the present invention is described in further detail below. Any polypeptide can be used in the screening methods of the invention as long as it has the biological activity of a SUV39H2 polypeptide. Examples of such biological activity include cell growth promoting activity and methyltransferase activity of SUV39H2 polypeptide. For example, SUV39H2 polypeptides can be used, and polypeptides functionally equivalent to SUV39H2 polypeptides can also be used. Such polypeptides may be expressed endogenously or exogenously by the cell. For details of the functional equivalents of SUV39H2 polypeptides, see the section “Genes and polypeptides”.

  The substance isolated by the screening method of the present invention is an antagonist (inhibitor) candidate for the SUV39H2 polypeptide. The term “antagonist” refers to a molecule that inhibits its function by binding to a polypeptide. The term also refers to a molecule that reduces or inhibits the expression of a gene encoding a SUV39H2 polypeptide. Moreover, substances isolated by the screening methods of the present invention are candidates for substances that inhibit in vivo interactions between SUV39H2 polypeptides and molecules (including DNA and proteins).

  When the biological activity to be detected in the method of the present invention is a cell growth promoting activity, it is possible to prepare cells expressing SUV39H2 polypeptide by, for example, MTT assay, colony formation assay or FACS, and The cells can be detected by culturing the cells in the presence, determining the rate of cell proliferation, measuring the cell cycle, and measuring cell survival or colony forming activity. A substance that reduces the growth rate of cells expressing the SUV39H2 gene is selected as a candidate substance for one or both of cancer treatment and prevention. In some embodiments, the cell expressing the SUV39H2 gene is an isolated and cultured cell that expresses the SUV39H2 gene exogenously or endogenously in vitro.

More specifically, the method includes the following steps:
(A) contacting the test substance with cells expressing the SUV39H2 gene;
(B) measuring cell growth promoting activity; and (c) selecting a test substance that reduces cell growth promoting activity compared to cell growth promoting activity in the absence of the test substance.
In a preferred embodiment, the method of the present invention may further comprise the following steps:
(D) selecting a test substance that has substantially no effect on cells that express little or no detectable SUV39H2 gene.

  When the biological activity to be detected in the method of the present invention is methyltransferase activity, the methyltransferase activity can be determined by using SUV39H2 polypeptide as a substrate (eg, histone H3, or a fragment thereof containing lysine 9 (eg, SEQ ID NO: 31 )) And a cofactor (eg, S-adenosyl-L-methionine) under conditions suitable for methylation of the substrate, and detecting the methylation level of the substrate.

More specifically, the present invention provides the following methods [1] to [7]:
[1] A method for screening a candidate substance for one or both of cancer treatment and prevention comprising the following steps:
(A) contacting a test substance with a SUV39H2 polypeptide or functional equivalent thereof, a substrate and a cofactor under conditions suitable for the methylation of the substrate;
(B) detecting the methylation level of the substrate; and (c) selecting a test substance that reduces the methylation level of the substrate compared to the methylation level in the absence of the test substance;
[2] The method according to [1] above, wherein the substrate is histone or a fragment thereof containing at least one methylation region;
[3] The method according to [2] above, wherein the substrate is histone H3 or a fragment thereof containing at least one methylation region;
[4] The method according to [3] above, wherein the methylation region is lysine 9 of histone H3;
[5] The method according to any one of [1] to [4], wherein the cofactor is S-adenosylmethionine;
[6] The method according to any one of [1] to [5] above, wherein the polypeptide is contacted with a substrate and a cofactor in the presence of an agent for enhancing methylation; and [7] Enhancement of methylation The method according to [6] above, wherein the agent is S-adenosylhomocysteine hydrolase (SAHH).

In the context of the present invention, the methyltransferase activity of a SUV39H2 polypeptide can be determined by methods known in the art (eg, WO01 / 094620, Hamamoto et al, Nature 2004; 6 (8): 731-740). O'carraol et al, Mol Cell Biol 2000; 20 (24): 9423-9433, Rea et al, Nature 2000; 406: 593-599).
For example, SUV39H2 polypeptide and substrate can be incubated with a labeled methylated donor under suitable assay conditions. Histone H3 peptide (i.e., histone H3 or fragment thereof), and S- adenosyl - [methyl ^- 14 C]-L-methionine or S- adenosyl, - a ^- [methyl 3 H]-L-methionine, preferably each It can be used as substrate and methyl donor. Transfer of the radiolabel to the histone H3 peptide can be detected by, for example, SDS-PAGE electrophoresis and fluorography. Alternatively, after the reaction, the histone H3 peptide can be separated from the methyl donor by filtration and the amount of radiolabel retained on the filter can be quantified by scintillation counting. Other suitable labels that can be attached to methyl donors are such as chromogenic labels and fluorescent labels, and methods for detecting the transfer of these labels to histones and histone peptides are known in the art. is there. An example of a methyltransferase assay is described in "Example 5: Screening for inhibitors of SUV39H2 methyltransferase activity".

  Alternatively, the methyltransferase activity of a SUV39H2 polypeptide is determined using a reagent that selectively recognizes an unlabeled methyl donor (eg, S-adenosyl-L-methionine) and a methylated substrate (eg, histone H3 peptide). Can be determined. For example, after incubation of the SUV39H2 polypeptide, the substrate to be methylated and the methyl donor under conditions that allow the substrate to be methylated, the methylated substrate can be detected by immunological methods. Any immunological technique using an antibody that recognizes a methylated substrate can be used for detection. For example, antibodies against methylated histones are commercially available (abcam Ltd.). ELISA or immunoblotting using antibodies that recognize methylated histones can be used for the present invention.

  In the context of the present invention, histone H3 or a fragment thereof (eg SEQ ID NO: 31) can preferably be used as a substrate to be methylated by SUV39H2 polypeptide. The histone H3 fragment to be used as a substrate preferably retains lysine 9. Such histone H3 fragments are preferably composed of at least 10 amino acid residues, more preferably at least 15 amino acid residues, and even more preferably at least 20 amino acid residues. An example of such a histone H3 fragment is a peptide having the amino acid sequence of SEQ ID NO: 31. Alternatively, a modified peptide of histone H3 or a fragment thereof such that methyltransferase has increased affinity / activity thereto may be used. Such peptides can be designed by exchanging and / or adding and / or deleting amino acids and testing the substrate in sequential experiments for methyltransferase assays using SUV39H2 polypeptides.

  In the context of the present invention, any functional equivalent of a SUV39H2 polypeptide can be used as long as it retains the methyltransferase activity of the original (native, wild type) SUV39H2 polypeptide. Thus, the functional equivalent of a SUV39H2 polypeptide preferably retains the SET-domain of the SUV39H2 polypeptide (see “Gene and Polypeptide” section). An example of such a functional equivalent is a polypeptide having the amino acid sequence of SEQ ID NO: 32.

  SUV39H2 polypeptide or a functional equivalent thereof is a fusion protein containing a monoclonal antibody recognition site (epitope) by introducing an epitope of a monoclonal antibody whose specificity has been elucidated into the N- or C-terminus of the polypeptide. It may be expressed. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors that can express fusion proteins with, for example, β-galactosidase, maltose-binding protein, glutathione-S-transferase (GST), green fluorescent protein (GFP), etc. by using multiple cloning sites are commercially available. Yes. A fusion protein prepared by introducing only a small epitope consisting of several to a dozen amino acids so as not to change the properties of the SUV39H2 polypeptide by fusion has also been reported. Polyhistidine (His-tag), influenza aggregated HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV- tag), E-tags (epitopes on monoclonal phage) and the like.

  The present invention contemplates the use of agents that enhance the methylation of the substance. A preferred enhancer for methylation is SAHH or a functional equivalent thereof. Such agents enhance the methylation of the substance, thereby enabling sensitive determination of methyltransferase activity. Thus, SUV39H2 may be contacted with the substrate and cofactor in the presence of an enhancer.

  Methyltransferase activity is determined by preparing cells expressing SUV39H2 polypeptide, culturing the cells in the presence of a test substance, and using, for example, an antibody that specifically binds to methylated histones to determine histone methylation levels. Can also be detected.

More specifically, the method includes the following steps:
[1] A step of bringing a test substance into contact with a cell expressing the SUV39H2 gene;
[2] detecting a methylation level of histone H3 lysine 9 (H3K9); and [3] selecting a test substance that reduces the methylation level compared to the methylation level in the absence of the test substance.

  As mentioned above, the phrase “suppressing (or inhibiting) biological activity” here refers to inhibition of the biological activity of the SUV39H2 polypeptide, preferably at least 10% compared to the absence of the substance. More preferably defined as at least 25%, 50% or 75% inhibition, and most preferably 90% inhibition. Thus, a test substance is characterized as “reducing the methylation level” if it provides a reduction on the order of 10%, more preferably at least a 25%, 50% or 75% reduction, and most preferably a 90% reduction. May be.

In a preferred embodiment, control cells that do not express the SUV39H2 gene are used. Accordingly, the present invention also provides a method for screening a candidate substance for inhibiting cell proliferation or a candidate substance for one or both of the treatment and prevention of SUV39H2-related cancer using SUV39H2 polypeptide or a functional equivalent thereof. There is provided a method comprising the following steps:
(A) culturing cells expressing the SUV39H2 polypeptide or functional equivalent thereof and control cells not expressing the SUV39H2 polypeptide or functional equivalent thereof in the presence of a test substance;
(B) detecting the biological activity (cell proliferation or methyltransferase activity) of cells expressing the SUV39H2 polypeptide or functional equivalent thereof and control cells; and (c) in the control cells and in the absence of the test substance. Biological activity (cell proliferation or methyltransferase activity) in cells expressing SUV39H2 polypeptide or a functional equivalent thereof compared to the biological activity detected below (cell proliferation promoting activity or methyltransferase activity) The step of selecting a test substance that inhibits

(IV) Screening for a substance that alters the expression of the SUV39H2 gene The present invention also provides a screening for a substance that inhibits the expression of the SUV39H2 gene. A substance that inhibits the expression of the SUV39H2 gene suppresses the growth of cancer cells (eg, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer or soft tissue tumor), and thus cancer (eg, It is expected to be useful for treatment and / or prevention of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer or soft tissue tumor). Therefore, the present invention also provides a method for screening a substance that suppresses the growth of cancer cells that overexpress the SUV39H2 gene, such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor cells, And screening methods for candidate substances for treatment and / or prevention of cancers associated with SUV39H2 overexpression, such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor To do.

In the context of the present invention, such a screening method can comprise, for example, the following steps:
(A) contacting the test substance with cells expressing the SUV39H2 gene;
(B) detecting the expression level of the SUV39H2 gene in the cell; and (c) selecting a test substance that reduces the expression level of the SUV39H2 gene compared to the expression level detected in the absence of the test substance. Process.

Furthermore, the present invention provides a method for evaluating or estimating the therapeutic effect of a test substance on suppression of cancer cell proliferation, or one or both of cancer treatment and prevention, and the method comprises the following steps: :
(A) contacting the substance with a cell expressing the SUV39H2 gene;
(B) detecting the expression level of the SUV39H2 gene; and (c) associating the potential therapeutic effect of the test substance with the expression level detected in step (b), wherein the potential therapeutic effect Is indicated when the test substance reduces the expression level of the SUV39H2 gene compared to the expression level detected in the absence of the test substance.

  In the context of the present invention, the therapeutic effect can be correlated with the expression level of the SUV39H2 gene. For example, if a test substance reduces the expression level of the SUV39H2 gene compared to the level detected in the absence of the test substance, the test substance may be identified or selected as a candidate substance having a therapeutic effect . Alternatively, if the test substance does not reduce the expression level of the SUV39H2 gene compared to the level detected in the absence of the test substance, the test substance may be identified as a substance that has no significant therapeutic effect. Good.

  Furthermore, downstream genes of the SUV39H2 gene that are affected by SUV39H2 knockdown were identified in the course of the present invention. Table 8 shows a list of genes that were down-regulated in SW780 and A549 cells transfected with SUV39H2 siRNA. Table 7 shows a list of genes that were up-regulated in SW780 and A549 cells transfected with SUV39H2 siRNA. The expression levels of these downstream genes can be used as an indicator of the expression level of the SUV39H2 gene.

Accordingly, the present invention also provides for the treatment and / or prevention of cancers associated with SUV39H2 overexpression (eg, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, canter or soft tissue tumor). For candidate or for prevention of proliferation of cancer cells overexpressing SUV39H2 (eg lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer or soft tissue tumor cells) A method is provided, the method comprising the following steps:
(A) contacting the test substance with a cell expressing a SUV39H2 gene and a downstream gene of the SUV39H2 gene selected from the genes shown in Tables 7 and 8;
(B) detecting the expression level of the downstream gene; and (c) selecting a test substance that changes the expression level of the downstream gene compared to the expression level detected in the absence of the test substance.

More specifically, when the downstream gene is a gene selected from the genes shown in Table 8, a test substance that reduces the expression level should be selected as a candidate substance. On the other hand, when the downstream gene is a gene selected from the genes shown in Table 7, a test substance that increases its expression level should be selected as a candidate substance. Therefore, in a preferred embodiment, the screening method of the present invention comprises the following steps:
(A) contacting the test substance with a cell expressing a downstream gene of the SUV39H2 gene selected from the SUV39H2 gene and the genes shown in Table 8;
(B) detecting the expression level of the downstream gene; and (c) selecting a test substance that reduces the expression level of the downstream gene compared to the expression level detected in the absence of the test substance.

Alternatively, in some embodiments, the invention is also a candidate for one or both of the treatment and prevention of cancer associated with SUV39H2 overexpression or for preventing the growth of cancer cells that overexpress SUV39H2. Also provided is a method of assessing or inferring the therapeutic effect of a substance, which method comprises the following steps:
(A) contacting the test substance with a cell expressing a downstream gene of the SUV39H2 gene selected from the SUV39H2 gene and the genes shown in Table 8;
(B) detecting the expression level of a downstream gene; and (c) associating a potential therapeutic effect with a test substance, wherein the potential therapeutic effect is compared to the control. Shown when reducing the expression level of downstream genes.

Alternatively, the screening method of the present invention may include the following steps:
(A) contacting the test substance with a cell expressing a downstream gene of the SUV39H2 gene selected from the SUV39H2 gene and the genes shown in Table 7;
(B) detecting the expression level of the downstream gene, and (c) selecting a test substance that increases the expression level of the downstream gene compared to the expression level detected in the absence of the test substance.

Alternatively, in some embodiments, the present invention provides a therapeutic effect of a candidate substance for the prevention and / or prevention of cancer cells associated with SUV39H2 overexpression, or for the prevention or growth of cancer cells that overexpress SUV39H2. Is also provided, and the method includes the following steps:
(A) contacting the test substance with a cell expressing a downstream gene of the SUV39H2 gene selected from the SUV39H2 gene and the genes shown in Table 7;
(B) detecting the expression level of a downstream gene, and (c) associating a potential therapeutic effect with a test substance, wherein the potential therapeutic effect is compared to the control. Shown when increasing the expression level of a downstream gene.

  The screening method of the present invention is described in further detail below. Cells expressing SUV39H2 and downstream genes shown in Tables 7 and 8 include, for example, cell lines established from lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer or soft tissue tumor It is done. Such cells (for example, SW780, RT4, A549, LC319, SBC5) can be used in the screening method of the present invention. Expression levels can be estimated by methods well known to those skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining and flow cytometry analysis. As described above, the phrase “reduces the expression level” here refers to an expression level of SUV39H2 or a downstream gene, preferably at least 10% compared to the expression level in the absence of the test substance. Reduced, more preferably defined as a level reduced by at least 25%, 50% or 75%, and most preferably reduced by 95%. Here, examples of the test substance include chemical substances and double-stranded molecules. Methods for preparing chemicals are described above. Methods for preparing double stranded molecules are described below. In the screening method of the present invention, the test substance that reduces the expression level of the SUV39H2 gene is a cancer associated with SUV39H2 overexpression, such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer, and soft tissue tumor. Can be selected as a candidate substance to be used for treatment or prevention. The potential of these candidate agents for treating or preventing cancer may be assessed by one or both of a second or additional screen to identify a therapeutic agent for cancer. In some embodiments, the cells that express the SUV39H2 gene are isolated and cultured cells that express the SUV39H2 gene exogenously or endogenously in vitro.

Alternatively, the screening method of the present invention may include the following steps:
(A) contacting the test substance with a cell into which a vector containing a transcriptional regulatory region of the SUV39H2 gene and a reporter gene expressed under the control of the transcriptional regulatory region has been introduced;
(B) detecting the expression level or activity of the reporter gene, and (c) a test substance that reduces the expression level or activity of the reporter gene compared to the expression level or activity detected in the absence of the test substance. Process to select.

Furthermore, the present invention provides a method for evaluating or estimating the therapeutic effect of a test substance on one or both of cancer treatment and prevention, or inhibition of cancer cell growth, and the method comprises the following steps: Including:
(A) contacting the test substance with a cell into which a vector containing a transcriptional regulatory region of the SUV39H2 gene and a reporter gene expressed under the control of the transcriptional regulatory region has been introduced;
(B) detecting the expression level or activity of the reporter gene, and (c) associating the potential therapeutic effect of the test substance with the expression level or activity detected in step (b), the potential therapeutic effect Is indicated when the test substance reduces the expression level or activity of the reporter gene compared to the expression level or activity detected in the absence of the test substance.

  In the context of the present invention, the therapeutic effect may be related to the expression level or activity of the reporter gene. For example, a test substance may be identified or selected as a candidate substance that has a therapeutic effect if the test substance reduces the expression level or activity of the reporter gene compared to the level detected in the absence of the test substance. . Alternatively, if the test substance does not reduce the expression level or activity of its reporter gene compared to the level detected in the absence of the test substance, the test substance is identified as a substance that has no significant therapeutic effect. May be.

  Suitable reporter genes and host cells are well known in the art. For example, reporter genes include luciferase, green fluorescent protein (GFP), anemone sp. Red fluorescent protein (DsRed), chloramphenicol acetyltransferase (CAT), lacZ and β-glucuronidase (GUS), and host The cells are COS7, HEK293, HeLa and the like. The reporter construct required for screening can be prepared by linking the reporter gene sequence to the transcriptional regulatory region of SUV39H2. Here, the transcriptional regulatory region of SUV39H2 is a region from the start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5000 or 10,000 bp upstream. Nucleotide segments containing transcriptional regulatory regions can be isolated from genomic libraries or can be amplified by PCR. The reporter construct required for screening can be prepared by linking the reporter gene sequence to the transcriptional regulatory region of any of these genes. Methods for identifying transcriptional regulatory regions and assay protocols are also well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).

  The vector containing the reporter construct infects the host cell, and reporter gene expression or activity is detected by methods well known in the art (eg, using a luminometer, absorptiometer, flow cytometer, etc.). In some embodiments, the cell of the invention is an isolated and cultured cell comprising a transcriptional regulatory region of the SUV39H2 gene and a reporter gene expressed under the control of the transcriptional regulatory region. Vectors to be introduced in vitro. As defined herein, “reducing expression or activity” preferably means at least a 10% reduction, more preferably at least 25%, of the expression or activity of a reporter gene compared to the absence of the substance, A 50% or 75% reduction, and most preferably a 95% reduction.

  Here, it is clarified that suppressing (reducing or inhibiting) the expression of the SUV39H2 gene suppresses (reduces or inhibits) cell proliferation. Therefore, by screening for candidate substances that reduce the expression or activity of the reporter gene, candidate substances having the potential to treat or prevent cancer can be identified. The potential of these candidate substances to treat or prevent cancer may be assessed by a second and / or further screen to identify cancer therapeutic substances.

Double-stranded molecule:
As used herein, the term “isolated double-stranded molecule” refers to a nucleic acid molecule that inhibits expression of a target gene, eg, small interfering RNA (siRNA; eg, double-stranded ribonucleic acid (dsRNA) Or small hairpin RNA (shRNA)) and small interfering DNA / RNA (siD / RNA; for example, DNA and RNA double-stranded chimeras (dsD / R-NA) or DNA and RNA small hairpin chimeras ( shD / R-NA)).
Here, “double-stranded molecule” means “double-stranded nucleic acid”, “double-stranded nucleic acid molecule”, “double-stranded polynucleotide”, “double-stranded polynucleotide molecule”, “double-stranded oligonucleotide”. And also referred to as “double-stranded oligonucleotide molecule”.

  As used herein, the term “target sequence” refers to a nucleotide sequence within the mRNA or cDNA sequence of a target gene, where a double-stranded molecule that targets that sequence is introduced into a cell that expresses that gene. Refers to those that result in the suppression of translation of the entire mRNA of the target gene. The nucleotide sequence within the mRNA or cDNA sequence of the target gene is determined to be the target sequence if a double-stranded molecule having a sequence corresponding to the target sequence inhibits the expression of the gene in a cell that expresses the gene be able to. Where the target sequence is represented by a cDNA sequence, the sense strand sequence of double stranded cDNA, ie, the sequence in which the mRNA sequence is converted to a DNA sequence, is used to define the target sequence. The double-stranded molecule is composed of a sense strand having a sequence corresponding to the target sequence and an antisense strand having a sequence complementary to the target sequence, and the antisense strand hybridizes with the sense strand in a complementary sequence. Forms a double-stranded molecule.

  Here, the phrase “corresponding to ()” means that the target sequence is converted according to the type of nucleic acid constituting the sense strand of the double-stranded molecule. For example, if the target sequence is indicated by a DNA sequence and the sense strand of a double-stranded molecule has an RNA region, base “t” in the RNA region is replaced with base “u”. On the other hand, when the target sequence is represented by an RNA sequence and the sense strand of the double-stranded molecule has a DNA region, the base “u” in the DNA region is replaced with “t”.

  For example, when the target sequence is the RNA sequence represented by SEQ ID NO: 19 or 20, and the sense strand of the double-stranded molecule has a 3 ′ half region composed of DNA, the “sequence corresponding to the target sequence” “5′-CUUUGGUUGTTCATGCCACA-3 ′” (for SEQ ID NO: 19), or “5′-CUGGAAUCAGCTTAGTCAA-3 ′” (for SEQ ID NO: 20).

  Moreover, the sequence complementary to the target sequence for the antisense strand of the double-stranded molecule can be defined according to the type of nucleic acid constituting the antisense strand. For example, when the target sequence is the RNA sequence shown in SEQ ID NO: 19 or 20, and the antisense strand of the double-stranded molecule has a 5 ′ half region composed of DNA, “the sequence complementary to the target sequence” Is “3′-GAAACCAACAAGTACGTGT-5 ′” (for SEQ ID NO: 19) or “3′-GACCUUAUGUCGAATCAGTT-5 ′” (for SEQ ID NO: 20).

  On the other hand, when the double-stranded molecule is composed of RNA, the sequence corresponding to the target sequence shown in SEQ ID NO: 19 or 20 is the RNA sequence of SEQ ID NO: 19 or 20, and the target sequence shown in SEQ ID NO: 19 or 20 The complementary sequence corresponding to is the RNA sequence of “3′-GAAACCAACAAGUACGUGU-5 ′” (for SEQ ID NO: 19) or “3′-GACCUUAUGUCGAAUCAGUU-5 ′” (for SEQ ID NO: 20).

  A double-stranded molecule is a loop sequence linking the sense and antisense strands to form one or two 3 ′ overhangs or hairpin structures having a length of 2-5 nucleotides (eg, uu), or Both may be present in addition to the sequence corresponding to the target sequence and the sequence complementary thereto.

  As used herein, the term “siRNA” refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including those in which DNA is the template from which RNA is transcribed. Alternatively, siRNA may be introduced directly into the cell to be treated. Methods for introducing siRNA into a subject are well known in the art. For example, administration of siRNA with a delivery agent is preferred for introduction of siRNA.

  The siRNA includes an SUV39H2 sense nucleic acid sequence (also referred to as “sense strand”), an SUV39H2 antisense nucleic acid sequence (also referred to as “antisense strand”) or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, eg, a hairpin. The siRNA may be either dsRNA or shRNA.

  As used herein, the term “dsRNA” refers to a construct of two RNA molecules that are composed of sequences that are complementary to each other and that anneal through the complementary sequences to form a double-stranded RNA molecule. The two-strand nucleotide sequence comprises not only “sense” or “antisense” RNA selected from the protein coding sequence of the target gene sequence, but also RNA molecules having a nucleotide sequence selected from the non-coding region of the target gene. May be included.

  The term “shRNA” as used herein refers to siRNA having a stem-loop structure composed of first and second regions, ie, sense and antisense strands, that are complementary to each other. The degree of complementarity and the orientation of the regions are sufficient for pairing between the regions to occur, the first and second regions being joined by a loop region, which loop is a nucleotide ( Or a lack of base pairing between nucleotide analogues). The loop region of shRNA is a single-stranded region interposed between the sense and antisense strands, and may be referred to as “intervening single-stranded”.

  As used herein, the term “siD / R-NA” refers to a double-stranded polynucleotide molecule composed of both RNA and DNA, including RNA and DNA hybrids and chimeras, and prevents translation of target mRNA. . Here, a hybrid refers to a molecule in which a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form a double-stranded molecule; whereas a chimera constitutes a double-stranded molecule Indicates that one or both strands may contain RNA and DNA. Standard techniques for introducing siD / RNA into cells are used. The siD / R-NA comprises a SUV39H2 sense nucleic acid sequence (also referred to as “sense strand”), a SUV39H2 antisense nucleic acid sequence (also referred to as “antisense strand”) or both. The siD / R-NA may be constructed, for example, like a hairpin, so that a single transcript has both sense and complementary antisense nucleic acid sequences from the target gene. siD / R-NA may be either dsD / R-NA or shD / R-NA.

  As used herein, the term “dsD / R-NA” is composed of sequences that are complementary to each other and anneals through those complementary sequences to form a double-stranded polynucleotide molecule. Refers to the construction of one molecule. A polynucleotide having a nucleotide sequence selected from a non-coding region of the target gene as well as a “sense” or “antisense” polynucleotide sequence selected from the protein coding sequence of the target gene sequence. May be included. One or both of the two molecules that make up dsD / R-NA may both be composed of RNA and DNA (chimeric molecules), or one molecule is composed of RNA and the other is composed of DNA. (Hybrid duplex).

  The term “shD / R-NA” as used herein refers to siD / R— having a stem-loop structure composed of first and second regions complementary to each other, ie, the sense and antisense strands. Refers to NA. The degree of complementarity and region orientation are sufficient for base pairing to occur between the regions, the first and second regions being joined by a loop region, and the loop is a nucleotide (or Resulting from the lack of base pairing between nucleotide analogues). The loop region of shD / R-NA is a single-stranded region interposed between the sense and antisense strands, and may also be referred to as “intervening single strand”.

  As used herein, an “isolated nucleic acid” is a nucleic acid that has been removed from its original environment (eg, the natural environment if it is naturally occurring), and thus has been altered synthetically from its natural state. ing. In the present invention, examples of isolated nucleic acids include DNA, RNA, and derivatives thereof.

In the context of the present invention, a double-stranded molecule to the SUV39H2 gene hybridizes to the target mRNA and associates with a transcript of this gene, usually a single-stranded mRNA, thereby interfering with the translation and thus of the protein. Inhibiting expression will reduce or inhibit production of the SUV39H2 protein encoded by the SUV39H2 gene. As revealed here, the expression of the SUV39H2 gene in several cancer cell lines is inhibited by dsRNA (FIGS. 4C and 4D). Thus, the present invention provides an isolated double-stranded molecule that can inhibit the expression of the SUV39H2 gene when introduced into a cell that expresses this gene. The target sequence of the double stranded molecule may be designed by an siRNA design algorithm as mentioned below.
Examples of the target sequence for the SUV39H2 gene include the nucleotide sequence of SEQ ID NO: 19 or 20.

Particularly interesting double-stranded molecules in the context of the present invention are defined in [1] to [18]:
[1] An isolated double-stranded molecule that, when introduced into a cell, inhibits in vivo expression and cell proliferation of the SUV39H2 gene, and is composed of a sense strand and an antisense strand complementary thereto, and hybridizes to each other. A molecule that forms a double-stranded molecule by soybeans;
[2] The double-stranded molecule according to [1], wherein the double-stranded molecule acts on mRNA and matches the target sequence of SEQ ID NO: 19 or 20;
[3] The double-stranded molecule according to [1], wherein the sense strand includes a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20;
[4] The double-stranded molecule according to any one of [1] to [3], which is less than about 100 nucleotides in length;
[5] The double-stranded molecule according to [4], which is less than about 75 nucleotides in length [6] The double-stranded molecule according to [5], which is less than about 50 nucleotides in length;
[7] The double-stranded molecule according to the above [6], which is less than about 25 nucleotides in length;
[8] The double-stranded molecule according to [7], which is about 19 to about 25 nucleotides in length;
[9] The double-stranded molecule according to any one of [1] to [8], comprising a single polynucleotide having both a sense and an antisense strand linked by an intervening single strand ;
[10] It has the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′, where [ A] is a sense strand comprising a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20; [B] is an intervening single strand composed of 3 to 23 nucleotides, and [A ′] is a target sequence The double-stranded molecule according to the above [9], which is an antisense strand comprising a sequence complementary to
[11] The double-stranded molecule according to any one of [1] to [10], which is composed of RNA;
[12] The double-stranded molecule according to any one of [1] to [10], which is composed of both DNA and RNA;
[13] The double-stranded molecule according to [12], wherein the molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[14] The double-stranded molecule according to the above [13], wherein the sense and antisense strands are respectively composed of DNA and RNA;
[15] The double-stranded molecule according to [12], wherein the molecule is a chimera of DNA and RNA;
[16] The region adjacent to the 3 ′ end of the antisense strand, or both the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are RNA [15] A double-stranded molecule according to
[17] The double-stranded molecule according to [16], wherein the adjacent region is composed of 9 to 13 nucleotides; and [18] The double-stranded according to [2], wherein the molecule contains a 3 ′ overhang. molecule;
The double-stranded molecules of the present invention are described in further detail below.

Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, eg, US Pat. No. 6,506,559, which is hereby incorporated by reference in its entirety). For example, a computer program for designing siRNA is available from the Ambion website (ambion.com/techlib/misc/siRNA_finder.html).
Such a computer program selects a target nucleotide sequence for a double-stranded molecule based on the following protocol.

Target site selection:
1. Starting from the AUG start codon of the transcript, the downstream is scanned for AA dinucleotide sequences. Record the occurrence of each AA and its 3 ′ adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. Recommends avoiding designing siRNAs for 5 'and 3' untranslated regions (UTRs) and regions close to the start codon (within 75 bases), as these are regulatory proteins This is because it may contain abundant binding sites and the UTR binding protein and / or translation initiation complex may interfere with the binding of the siRNA endonuclease complex.
2. Potential target sites are compared to the appropriate genomic database (human, mouse, rat, etc.) and any target sequences that have significant homology to other coding sequences are excluded from consideration. BLAST, which can be found on the NCBI server at ncbi.nlm.nih.gov/BLAST/, is used (Altschul SF et al., Nucleic Acids Res 1997 Sep 1, 25 (17): 3389-402).
3. Eligible target sequences are selected for synthesis. Typically, several target sequences are selected along the length of the gene to be evaluated.

  Using the above protocol, the target sequence of the isolated double-stranded molecule of the present invention was designed as SEQ ID NO: 19 or 20. Double-stranded molecules targeting the target sequences referred to above were each tested for their ability to inhibit the growth of cells expressing the target gene. Accordingly, the present invention provides a double-stranded molecule targeting the sequence of SEQ ID NO: 19 or 20 for the SUV39H2 gene.

  Examples of double-stranded molecules of the invention that target the target sequence of the SUV39H2 gene include isolated nucleic acid sequences corresponding to either the target sequence or a sequence complementary to the target sequence, or both A polynucleotide is mentioned. Preferable examples of the polynucleotide targeting the SUV39H2 gene include a sequence corresponding to SEQ ID NO: 19 or 20, and / or a sequence complementary to these sequences. In some embodiments, the double-stranded molecule is composed of two polynucleotides, one polynucleotide having a target sequence, i.e., a sequence corresponding to the sense strand, and the other polypeptide is relative to the target sequence. It has a complementary sequence, ie the antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form a double stranded molecule. Examples of such double stranded molecules include dsRNA and dsD / R-NA.

  In another embodiment, the double-stranded molecule is composed of a polynucleotide having both a target sequence, ie, a sequence corresponding to the sense strand, and a sequence complementary to the target sequence, ie, the antisense strand. In general, the sense strand and the antisense strand are connected by an intervening strand and hybridize with each other to form a hairpin loop structure. Examples of such double-stranded molecules include shRNA and shD / R-NA.

  In other words, the double-stranded molecule of the present invention is composed of a sense strand polynucleotide having a nucleotide sequence of a target sequence and an antisense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both polynucleotides are mutually Hybridizes to form double stranded molecules. In a double-stranded molecule comprising this polynucleotide, part of one or both polynucleotides of the strand may be RNA, and when the target sequence is defined by a DNA sequence, the target sequence and its complementary sequence Nucleotide “t” within is replaced by “u”.

In one embodiment of the invention, such a double stranded molecule of the invention comprises a stem loop structure composed of a sense and an antisense strand. The sense strand and the antisense strand may be connected by a loop. Thus, the present invention also provides a double stranded molecule composed of a single polynucleotide comprising both a sense strand and an antisense strand joined or flanked by intervening single strands.
The double-stranded molecule of the present invention may be directed to a single target SUV39H2 gene sequence or may be directed to multiple target SUV39H2 gene sequences.

  The double-stranded molecule of the present invention targeting the above targeting sequence of the SUV39H2 gene comprises an isolated polynucleotide comprising a target sequence and / or a nucleic acid sequence of a sequence complementary to the target sequence. Examples of polynucleotides that target the SUV39H2 gene include the sequences of SEQ ID NO: 19 or 20, and / or sequences complementary to these nucleotides. However, the present invention is not limited to this example, and minor modifications in the nucleic acid sequence described above are acceptable as long as the modified molecule retains the ability to suppress the expression of the SUV39H2 gene. Here, the phrase “minor modification” as used in connection with a nucleic acid sequence indicates one, two or several substitutions, deletions, additions or insertions of the nucleic acid to the sequence. In the context of the present invention, the term “several” applied to nucleic acid substitutions, deletions, additions and / or insertions is 3-7, preferably 3-5, more preferably 3-4, More preferably it can mean 3 nucleic acid residues.

  According to the present invention, the double-stranded molecules of the present invention can be tested for the ability to reduce, suppress or inhibit expression using the methods used in the Examples. Here, in the following examples, double-stranded molecules composed of sense strands of various parts of mRNA of SUV39H2 gene or antisense strands complementary thereto are expressed in cancer cell lines according to standard methods. It was tested in vitro for its ability to reduce the production of SUV39H2 gene product. Further, for example, the reduction of SUV39H2 gene product in cells contacted with a candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be performed, for example, as described in Example 1, “Quantitative RT-PCR. ”, Which can be detected by RT-PCR using primers for SUV39H2 mRNA. Sequences that reduce the production of SUV39H2 gene products in in vitro cell-based assays can then be tested for their inhibitory effect on cell proliferation. Sequences that inhibit cell proliferation in an in vitro cell-based sequence assay were then tested for their in vivo ability using animals with cancer, such as a nude mouse graft model, although reduced production and reduced production of SUV39H2 products. Can confirm cell proliferation.

  If the isolated polynucleotide is RNA or a derivative thereof, the base “t” should be replaced with “u” in the nucleotide sequence. As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a polynucleotide, and the term “binds” refers to two polynucleotides. Means physical or chemical interaction between them. If the polynucleotide comprises modified nucleotides and / or non-phosphodiester linkages, these polynucleotides can be linked together in the same way. In general, complementary polynucleotide sequences will hybridize under appropriate conditions to form a stable duplex with little or no mismatch. However, the present invention extends to complementary sequences containing one or more nucleotide mismatches. Furthermore, the sense strand and the antisense strand of the isolated polynucleotide of the present invention can form a double-stranded molecule or a hairpin loop structure by hybridization. In preferred embodiments, such duplexes do not contain more than 1 mismatch per 10 matches. In particularly preferred embodiments, such duplexes do not contain mismatches if the strands of the duplex are sufficiently complementary.

  The complementary or antisense polynucleotide is preferably less than 1,000 nucleotides in length for the SUV39H2 gene. Preferably, the polynucleotide is less than 500, 200, 100, 75, 50, or 25 nucleotides in length for all of the genes. The isolated polynucleotides of the present invention are useful for the formation of double stranded molecules against the SUV39H2 gene or for the preparation of template DNA encoding double stranded molecules. When the polynucleotide is used to form a double-stranded molecule, the sense strand of the polynucleotide may be longer than 19 nucleotides, preferably longer than 21 nucleotides, more preferably about 19-25 nucleotides in length. .

  Thus, the present invention provides a double stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to the target sequence. In a preferred embodiment, the sense strand hybridizes with the antisense strand at the target sequence to form a double stranded molecule having a length of 19-25 nucleotide pairs.

  The double-stranded molecule serves as a guide for identifying homologous sequences in the mRNA for the RISC complex when the double-stranded molecule is introduced into the cell. The identified target RNA is cleaved and degraded by Dicer's nuclease activity, through which double-stranded molecules ultimately reduce or inhibit production (expression) of the polypeptide encoded by the RNA. Accordingly, the double-stranded molecule of the present invention can be defined by the ability to generate a single strand that specifically hybridizes to the mRNA of the SUV39H2 gene under stringent conditions. Here, the part of mRNA that hybridizes with a single strand generated from a double-stranded molecule is called a “target sequence” or “target nucleic acid” or “target nucleotide”. In the present invention, the nucleotide sequence of the “target sequence” can be shown using not only the RNA RNA sequence but also the DNA sequence of cDNA synthesized from the mRNA.

  Alternatively, the double-stranded molecule of the present invention has a sense strand that hybridizes with the antisense strand at the target sequence to form a double-stranded molecule having a length of less than 500, 200, 100, 75, 50, or 25 nucleotide pairs. It may be a double-stranded molecule. Preferably, the double-stranded molecule has a length of about 19 to about 25 nucleotide pairs. Furthermore, the sense strand of the double-stranded molecule preferably comprises less than 500, 200, 100, 75, 50, 30, 28, 27, 26, 25 nucleotides, more preferably from about 19 to about 25 nucleotides.

  The double-stranded molecules of the present invention may contain one or more modified nucleotides and / or non-phosphodiester linkages. It is well known in the art to introduce chemical modifications that can increase the stability, availability and / or cellular uptake of double-stranded molecules. The person skilled in the art knows a wide range of chemical modifications that can be incorporated into the molecules of the invention (WO03 / 070744; WO2005 / 045037). For example, in one embodiment, the modification can be used to provide improved degradation resistance or improved uptake. Examples of such modifications include phosphorothioate linkages, 2′-O-methylribonucleotides (especially on the sense strand of double-stranded molecules), 2′-deoxy-fluororibonucleotides, 2′-deoxyribonucleotides, “universal bases” ”Nucleotides, 5′-C-methyl nucleotides, and reverse deoxybasic residue incorporation (US200602122137).

  In another embodiment, the modifications can be used to increase the targeting efficiency or enhance the stability of the double-stranded molecule. Examples of such modifications include chemical cross-linking between two complementary strands of a double-stranded molecule, chemical modification of the 3 ′ or 5 ′ end of one strand of a double-stranded molecule, sugar modification, nucleobase modification, and Examples include / but are not limited to backbone modifications, 2-fluoro modified ribonucleotides and 2′-deoxy ribonucleotides (WO 2004/029212). In another aspect, the modifications can be used to increase or decrease the affinity for complementary nucleotides in the target mRNA and / or complementary double stranded molecular strands (WO 2005/044976). For example, unmodified pyrimidine nucleotides can be replaced with 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Furthermore, the unmodified purine can be substituted with 7-deaza, 7-alkyl, or 7-alkenylpurine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3 ′ overhang, the overhanging nucleotide of the 3 ′ terminal nucleotide may be replaced with deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15 (2): 188-200).

  For further details, see for example US20060234970. However, the invention should not be limited as being limited to these examples; any of a number of chemical modifications can be used as long as the resulting molecule retains the ability to inhibit the expression of the target gene. It can be used for the double-stranded molecule of the present invention.

  The double-stranded molecule of the present invention may include both DNA and RNA, for example dsD / R-NA or shD / R-NA. For example, DNA strand and RNA strand hybrid polynucleotides or DNA-RNA chimeric polynucleotides exhibit increased stability and are therefore contemplated herein. A mixture of DNA and RNA, ie, a hybrid type double-stranded molecule composed of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), both DNA and RNA being either single-stranded (polynucleotide) or Chimeric-type double-stranded molecules and the like included in both may be formed in order to enhance the stability of the double-stranded molecule.

  DNA strand and RNA strand hybrids were coupled to the RNA antisense strand as long as the resulting double-stranded molecule was able to inhibit expression of the target gene when introduced into a cell expressing the gene. It may have a DNA sense strand, or vice versa. In a preferred embodiment, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.

  Chimeric-type double-stranded molecules are composed of DNA and RNA as long as the resulting double-stranded molecule has the activity of inhibiting the expression of the target gene when introduced into a cell that expresses the gene. One or both of the sense and antisense strands may be included, or any one of the sense and antisense strands composed of DNA and RNA may be included. In order to enhance the stability of the double-stranded molecule, this molecule preferably contains as much DNA as possible, while in order to induce inhibition of target gene expression, this molecule induces sufficient inhibition of expression. It is required to be RNA to the extent that it does.

A preferred chimeric type of double-stranded molecule comprises an upstream partial region of RNA (ie, a region adjacent to a target sequence or its complementary sequence in the sense or antisense strand). Preferably, the upstream partial region indicates the 5 ′ side (5 ′ end) of the sense strand and the 3 ′ side (3 ′ end) of the antisense strand. Alternatively, the region adjacent to the 5 ′ end of the sense strand and / or the 3 ′ end of the antisense strand may be referred to as the upstream partial region. That is, in a preferred embodiment, the region adjacent to the 3 ′ end of the antisense strand, or both the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are composed of RNA. The For example, a chimeric or hybrid type double-stranded molecule of the invention may comprise the following combinations:
Sense strand:
5 '-[--- DNA ---]-3'
3 ′-(RNA)-[DNA] -5 ′
: Antisense strand,
Sense strand:
5 ′-(RNA)-[DNA] -3 ′
3 ′-(RNA)-[DNA] -5 ′
: Antisense strand and sense strand:
5 ′-(RNA)-[DNA] -3 ′
3 '-(--- RNA ---)-5'
: Antisense strand.

  The upstream partial region is preferably a domain composed of 9 to 13 nucleotides from the end of the target sequence in the sense or antisense strand of the double-stranded molecule or a sequence complementary thereto. Moreover, as a preferable example of such a chimeric type double-stranded molecule, it has a chain of 19 to 21 nucleotides in length, and is at least an upstream half region (a 5 ′ region and an antisense region with respect to the sense strand). Examples include those in which the 3 ′ region of the strand is RNA and the other half is DNA. In such a chimeric-type double-stranded molecule, the effect of inhibiting the expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).

  In the context of the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and a short hairpin composed of DNA and RNA (shD / R-NA). shRNA or shD / R-NA is a sequence of RNA or a mixture of RNA and DNA that creates a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD / R-NA comprises a sense target sequence and an antisense target sequence on a single strand, where these sequences are separated by a loop sequence. In general, the hairpin structure is cleaved by a cellular device into dsRNA or dsD / R-NA molecules, which then bind to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the target sequence of dsRNA or dsD / R-NA.

  To form a hairpin loop structure, a loop sequence composed of any nucleotide sequence can be located between the sense and antisense sequences. Such a loop sequence may be linked to the 5 'or 3' end of the sense strand to form a hairpin loop structure. Accordingly, the present invention provides a duplex having the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′. A molecule wherein [A] is a sense strand comprising a sequence corresponding to the target sequence, [B] is an intervening single strand, and [A ′] is complementary to [A] Also provided are double stranded molecules, which are antisense strands comprising sequences. The target sequence may be selected, for example, from the nucleotide sequence of SEQ ID NO: 19 or 20.

  The present invention is not limited to these examples, and the target sequence in [A] is modified from these examples as long as the double-stranded molecule retains the ability to suppress the expression of the target SUV39H2 gene. It may be an array. Region [A] hybridizes to [A ′] to form a loop composed of region [B]. The intervening single-stranded portion [B], ie the loop sequence, can preferably be 3 to 23 nucleotides in length. The loop sequence can be selected, for example, from sequences found on the Ambion website (ambion.com/techlib/tb/tb_506.html). Furthermore, a loop sequence composed of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418 (6896): 435-8, Epub 2002 Jun 26): CCC, CCACC, Or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418 (6896): 435-8, Epub 2002 Jun 26; UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20 (5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100 (4): 1639-44, Epub 2003 Feb 10; and UUCAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4 (6) : 457-67. )

Examples of preferred double-stranded molecules of the present invention having a hairpin loop structure are shown below. In the following structure, the loop sequence can be selected from AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGGA; however, the present invention is not limited thereto:
CUUGUGGUUGUCAUCGCACA- [B] -UGUGCAUGAACACACAAAG (SEQ ID NO: 19 for target sequence) and CUGGAAUCAGCUUAUGUCAA- [B] -UUGACUAAGCUGAUUCCAG (SEQ ID NO: 20 for target sequence).

  In addition, some nucleotides can be added as 3 'overhangs to the 3' end of the sense and / or antisense strand of the target sequence to enhance the inhibitory activity of the double stranded molecule. Preferred examples of nucleotides making up the 3 'overhang include, but are not limited to, "t" and "u". The number of nucleotides to be added is at least 2, generally 2-10, preferably 2-5. The added nucleotides form a single strand at the 3 'end of the sense and / or antisense strand of the double-stranded molecule. If the double stranded molecule is composed of a single polynucleotide to form a hairpin loop structure, a 3 'overhang sequence may be added to the 3' end of the single polynucleotide.

  The method for preparing the double-stranded molecule is not particularly limited, but it is preferable to use any of the standard chemical methods known in the art. According to one chemical synthesis method, sense and antisense single stranded polynucleotides may be synthesized separately and then annealed to each other via an appropriate method to yield a double stranded molecule. In one specific embodiment, the synthesized single-stranded polynucleotide is preferably at least about 3: 7, more preferably about 4: 6 molar ratio, and most preferably substantially equimolar amounts (ie, , About 5: 5 molar ratio). The mixture is then heated to a temperature at which the double-stranded molecules dissociate and then gradually cooled. The annealed double-stranded polynucleotide can be purified by a known method usually used in the art. Examples of purification methods include a method using agarose gel electrophoresis or a method in which the remaining single-stranded polynucleotide is optionally removed, for example, by degradation using a suitable enzyme.

  The regulatory sequences adjacent to the SUV39H2 sequence may be the same or different so that their expression can be regulated independently or in a temporal or spatial manner. Double-stranded molecules can be transcribed intracellularly, for example, by cloning the SUV39H2 gene template into a vector containing a small nuclear RNA (snRNA) U6-derived RNA pol III transcription unit or a human H1 RNA promoter.

Vector encoding the double-stranded molecule of the invention:
Also included in the present invention are vectors encoding one or more double-stranded molecules described herein, and cells containing such vectors. Specifically, the present invention provides the following vectors [1] to [10].
[1] When introduced into a cell, encodes a double-stranded molecule that inhibits in vivo expression and cell proliferation of the SUV39H2 gene, and this molecule is composed of a sense strand and a complementary antisense strand, and hybridizes to each other To form a double-stranded molecule.
[2] The vector according to [1], which encodes a double-stranded molecule acting on mRNA that matches the target sequence of SEQ ID NO: 19 or 20.
[3] The vector according to [1], wherein the sense strand includes a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20.
[4] The vector according to [3], which encodes a double-stranded molecule having a length of less than about 100 nucleotides;
[5] The vector according to [4], which encodes a double-stranded molecule having a length of less than about 75 nucleotides;
[6] The vector according to [5], which encodes a double-stranded molecule having a length of less than about 50 nucleotides;
[7] The vector according to [6], which encodes a double-stranded molecule having a length of less than about 25 nucleotides;
[8] The vector according to [7], which encodes a double-stranded molecule having a length of about 19 to about 25 nucleotides;
[9] The double-stranded molecule according to any one of the above [1] to [8], wherein the double-stranded molecule is composed of a single polynucleotide having both a sense and an antisense strand linked by an intervening single strand. The described vectors;
[10] Encodes a double-stranded molecule having the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′ Here, [A] is a sense strand comprising a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, [B] is an intervening single strand composed of 3 to 23 nucleotides, and [ A ′] is the antisense strand containing a sequence complementary to the target sequence, the vector according to [9] above.

  The vector of the present invention preferably encodes the double-stranded molecule of the present invention in an expressible form. Here, the phrase “in expressible form” indicates that the vector, when introduced into a cell, will express a molecule carried, contained or encoded therein. In a preferred embodiment, the vector contains one or more regulatory elements necessary for expression of the double-stranded molecule. Thus, in one embodiment, the expression vector encodes a nucleic acid sequence of the invention and is adapted for expression of that nucleic acid sequence. Such vectors of the present invention may be used directly to generate the double-stranded molecule of the present invention or directly as an active ingredient for treating cancer.

  The vector of the present invention clones the SUV39H2 sequence into the expression vector so that the regulatory sequence is operably linked to the SUV39H2 sequence, for example, in a manner that allows expression of both strands (by transcription of the DNA molecule). (Lee NS et al., Nat Biotechnol 2002 May, 20 (5): 500-5). For example, an RNA molecule that is antisense to mRNA is transcribed by a first promoter (eg, a promoter sequence adjacent to the 3 ′ end of the cloned DNA), and an RNA molecule that is sense strand to mRNA is , Transcribed by a second promoter (eg, a promoter sequence adjacent to the 5 ′ end of the cloned DNA). The sense and antisense strands hybridize in vivo to produce a double stranded molecular construct for gene silencing. Alternatively, two vector constructs that encode the sense and antisense strands of the double-stranded molecule, respectively, are utilized to express the sense and antisense strands, respectively, to form a double-stranded molecule construct. In addition, the cloned sequence may encode a construct with secondary structure (eg, a hairpin); thus, a single transcript of the vector will contain both the sense and complementary antisense sequences of the target gene. May be included.

  The present invention is a vector comprising each or both of a combination of polynucleotides, including a sense strand nucleic acid and an antisense strand nucleic acid, wherein the antisense strand comprises a nucleotide sequence complementary to the sense strand, A transcript that hybridizes to each other to form a double-stranded molecule, and the vector inhibits expression of the gene when introduced into a cell that expresses the SUV39H2 gene Contemplate.

  The vectors of the present invention may be equipped to achieve stable insertion into the genome of the target cell (see, eg, Thomas KR & Capecchi MR, Cell 1987, 51: 503 for a description of homologous recombination cassette vectors). (See -12). For example, Wolff et al., Science 1990, 247: 1465-8; US Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; See, 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery techniques include “naked DNA”, facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (eg, U.S. Pat. No. 5,922,687).

  Examples of the vector of the present invention include viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia or fowlpox virus (see, eg, US Pat. No. 4,722,848). This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences encoding double-stranded molecules. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses this molecule, thereby inhibiting cell growth. Another example of a vector that can be used is Bacillus Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A great variety of other vectors are useful for the generation and therapeutic administration of double-stranded molecules; examples include adeno and adeno-associated viral vectors, retroviral vectors, Salmonella typhi vectors, detoxification And an anthrax toxin vector. For example, Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85. Please refer.

Cancer treatment method using the double-stranded molecule of the present invention:
In the present invention, dsRNAs against the SUV39H2 gene were tested for their ability to inhibit cell proliferation. DsRNA against the SUV39H2 gene efficiently knocked down gene expression in several cancer cell lines and coincided with suppression of cell proliferation (FIG. 4).

  Accordingly, the present invention provides a method of inhibiting the growth of cells, ie, cancer cells, by inducing dysfunction of the SUV37H2 gene through inhibition of SUV39H2 gene expression. SUV39H2 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention that specifically target the SUV39H2 gene.

  Such ability of the double-stranded molecules and vectors of the present invention to inhibit cell growth of cancerous cells allows them to treat cancer as well as its recurrence after surgery, secondary or metastatic. Or can be used for preventive methods. Exemplary cancers include, but are not limited to, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer, and soft tissue tumor, for example. Thus, the present invention treats patients with cancer without side effects because the SUV39H2 gene is detected in minimal amounts in normal organs by administering a double-stranded molecule to the SUV39H2 gene or a vector expressing this molecule. A method is provided (FIG. 1).

Of particular interest in the present invention are the methods [1] to [32] defined below:
[1] A method of inhibiting the growth of cancer cells or treating cancer, wherein the cancer cells or cancer express a SUV39H2 gene, wherein the method comprises a single method against at least one SUV39H2 gene. Administering a released double-stranded molecule or a vector encoding the double-stranded molecule, wherein the double-stranded molecule inhibits expression of this gene and cell proliferation in cells overexpressing the SUV39H2 gene Wherein the double-stranded molecule is composed of a sense strand and a complementary antisense strand, and hybridizes to each other to form a double-stranded molecule;
[2] The method according to [1] above, wherein the double-stranded molecule acts on mRNA corresponding to the target sequence of SEQ ID NO: 19 or 20.
[3] The method according to [1] above, wherein the sense strand comprises a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20.
[4] Any of the above-mentioned [1] to [3], wherein the cancer to be treated is selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor The method according to paragraph 1;
[5] The method according to any one of [1] to [4] above, wherein the double-stranded molecule is less than about 100 nucleotides in length;
[6] The method according to [5] above, wherein the double-stranded molecule is less than about 75 nucleotides in length;
[7] The method according to [6] above, wherein the double-stranded molecule is less than about 50 nucleotides in length;
[8] The method according to [7] above, wherein the double-stranded molecule is less than about 25 nucleotides in length;
[9] The method according to [8] above, wherein the double-stranded molecule has a length of about 19 to less than about 25 nucleotides;
[10] Any one of the above [1] to [9], wherein the double-stranded molecule is composed of a single polynucleotide comprising both a sense strand and an antisense strand linked by an intervening single strand. The method described in;
[11] The double-stranded molecule has the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′ Wherein [A] is a sense strand comprising a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, [B] is an intervening single strand composed of 3 to 23 nucleotides, and [ A ′] is an antisense strand comprising a sequence complementary to the target sequence, the method according to [10] above;
[12] The method according to any one of [1] to [11] above, wherein the double-stranded molecule is composed of RNA;
[13] The method according to any one of [1] to [12] above, wherein the double-stranded molecule is composed of both DNA and RNA;
[14] The method according to [13] above, wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[15] The method according to [14] above, wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[16] The method according to [13] above, wherein the double-stranded molecule is a DNA and RNA chimera;
[17] The region adjacent to the 3 ′ end of the antisense strand, or both the region adjacent to the 5 ′ end of the sense strand and the region adjacent to the 3 ′ end of the antisense strand are composed of RNA. 16];
[18] The method according to [17] above, wherein the adjacent region is composed of 9 to 13 nucleotides;
[19] The method according to any one of [1] to [18] above, wherein the double-stranded molecule comprises a 3 ′ overhang;
[20] The method according to any one of [1] to [19] above, wherein the double-stranded molecule is contained in a composition comprising a transfection enhancing agent and a pharmaceutically acceptable carrier;
[21] The method according to any one of [1] to [20] above, wherein the double-stranded molecule is encoded by a vector;
[22] The method according to [21] above, wherein the double-stranded molecule encoded by the vector acts on mRNA corresponding to the target sequence of SEQ ID NO: 19 or 20.
[23] The method according to [21] above, wherein the sense strand of the double-stranded molecule encoded by the vector comprises a sequence corresponding to a target sequence selected from SEQ ID NO: 19 or 20.
[24] Any one of [21] to [23] above, wherein the cancer to be treated is lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer (canter), or soft tissue tumor. The method described in;
[25] The method according to any one of [21] to [24] above, wherein the double-stranded molecule encoded by the vector is less than about 100 nucleotides in length;
[26] The method described in [25] above, wherein the double-stranded molecule encoded by the vector is less than about 75 nucleotides in length;
[27] The method described in [26] above, wherein the double-stranded molecule encoded by the vector is less than about 50 nucleotides in length;
[28] The method according to [27] above, wherein the double-stranded molecule encoded by the vector is less than about 25 nucleotides in length;
[29] The method according to [28] above, wherein the double-stranded molecule encoded by the vector is about 19 to about 25 nucleotides in length;
[30] The above [21] to [29], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide comprising both a sense strand and an antisense strand linked by an intervening single strand. The method according to any one of
[31] The double-stranded molecule encoded by the vector has the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′, wherein [A] is a sense strand comprising a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, and [B] is an intervening single strand composed of 3 to 23 nucleotides And [A ′] is an antisense strand comprising a sequence complementary to the target sequence; and [32] the vector is a transfection enhancing agent and a pharmaceutically acceptable The method according to [21] above, which is contained in a composition containing a possible carrier.

  The treatment and prevention methods of the present invention are described in detail below. Growth of cells that express the SUV39H2 gene may be inhibited by contacting the cells with a double-stranded molecule for the SUV39H2 gene, a vector that expresses this molecule, or a composition comprising the same. The cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase “inhibition of cell proliferation” indicates that the cell grows at a slower rate or has reduced viability compared to cells not exposed to the molecule. Cell proliferation can be measured by any number of methods known in the art, for example, using a colony formation assay or an MTT cell proliferation assay.

  As long as the cell expresses or overexpresses the target gene of the double-stranded molecule of the present invention, the growth of any kind of cell can be suppressed by the method of the present invention. Exemplary cells include lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor.

  Thus, a patient suffering from or at risk of developing a disease associated with SUV39H2 overexpression may comprise a double-stranded molecule of the invention, at least one vector expressing the molecule or a composition comprising the molecule. Treatment may be by administration. For example, cancer patients may be treated according to the methods of the present invention. The type of cancer may be identified by standard methods according to the specific type of tumor to be diagnosed. More preferably, patients to be treated by the methods of the invention are selected by detecting the expression of the SUV39H2 gene in a biopsy sample from the patient by RT-PCR or immunoassay. Preferably, prior to the treatment of the present invention, a biopsy specimen from a subject is confirmed for SUV39H2 gene overexpression by methods known in the art, such as immunohistochemical analysis or RT-PCR.

  In order to inhibit cell growth, the double-stranded molecule of the invention may be introduced directly into the cell in a form to achieve binding of this molecule to the corresponding mRNA transcript. Alternatively, as described above, DNA encoding a double-stranded molecule may be introduced into a cell by means of a vector. To introduce double-stranded molecules and vectors into cells, transfection enhancing agents such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleo Nucleofector (Wako pure Chemical) may be used.

  As described in the “Definitions” section above, treatment results in clinical efficacy such as reduced expression of the SUV39H2 gene or reduced cancer size, morbidity, or metastatic potential in the subject. In some cases, it is recognized as “effective”. “Effective” when treatment is applied prophylactically means delaying or preventing the formation of cancer, or preventing or alleviating the clinical symptoms of cancer. Efficacy is determined in relation to any known method for diagnosing or treating a particular tumor type.

  One skilled in the art understands that the double-stranded molecules of the present invention degrade SUV39H2 mRNA in substoichiometric amounts. While not wishing to be bound by any theory, it is believed that the double-stranded molecule of the present invention causes target mRNA degradation in a catalytic manner. Therefore, significantly fewer double-stranded molecules compared to standard cancer therapy need to be delivered to or near the cancer site in order to exhibit a therapeutic effect.

  The skilled artisan will determine the optimal effective amount of the double-stranded molecule of the invention to be administered to a given subject, such as the subject's weight, age, sex, disease type, symptom and other conditions; Can be readily determined by taking into account factors such as whether the administration is local or systemic; In general, an effective amount of a double-stranded molecule of the present invention is about 1 nanomolar (nM) to about 100 nM, preferably about 2 nM to about 50 nM, more preferably about 2.5 nM to about at or near the cancer site. 10 nM intercellular concentration. It is contemplated that lower or higher amounts of double-stranded molecules can be administered. The exact dosage required for a particular situation can readily be routinely determined by one skilled in the art.

The methods of the present invention can be used to grow or metastasize cancers that express the SUV39H2 gene, and more specifically overexpressed cancers such as lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumors. Can be used to inhibit.
In particular, a double-stranded molecule comprising the target sequence of the SUV39H2 gene (ie SEQ ID NO: 19 or 20) is particularly preferred for the treatment of cancer.

  In order to treat cancer, the double-stranded molecule of the present invention can be administered to a subject in combination with a pharmaceutical substance different from the double-stranded molecule. Alternatively, the double-stranded molecule of the present invention can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, the double-stranded molecules of the invention can be used to treat therapeutic methods currently used for cancer treatment or prevention of cancer metastasis (eg, radiation therapy, surgery and chemotherapeutic agents (eg, cisplatin, carboplatin, It can be administered in combination with treatment with cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen)). .

In the context of the present invention, the double-stranded molecule can be administered to the subject as a naked double-stranded molecule in combination with a delivery reagent or as a recombinant plasmid or viral vector that expresses the double-stranded molecule.
Suitable delivery reagents for administration in combination with the double-stranded molecules of the present invention include Milus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or poly Examples include cations or polycations (eg, polylysine), or liposomes. A preferred delivery reagent is a liposome.

  Liposomes can assist in the delivery of double-stranded molecules to specific tissues such as lung tumor tissue, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumors. The blood half-life of can also be increased. Liposomes suitable for use in the present invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and sterols such as cholesterol. The choice of lipid is generally guided by consideration of factors such as the desired liposome size and the half-life of the liposomes in the bloodstream. A variety of methods are known for the preparation of liposomes, eg, Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728. 4,837,028; and 5,019,369, the entire disclosures of which are incorporated herein by reference.

  Preferably, the liposome encapsulating the double-stranded molecule of the present invention includes a ligand molecule that can deliver the liposome to a cancer site. Preference is given to ligands that bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens.

  Particularly preferably, the liposomes encapsulating the double-stranded molecule of the invention are modified to avoid clearance by mononuclear macrophages and the reticuloendothelial system, for example by having an opsonization-inhibiting moiety attached to the surface of the structure Is done. In one embodiment, the liposomes of the invention can include both an opsonization inhibiting moiety and a ligand.

  Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that bind to the liposome membrane. As used herein, an opsonization-inhibiting moiety is chemically or physically attached to it, for example, by intercalation of a lipophilic anchor to the membrane itself or by direct attachment to the active group of the membrane lipid. When attached to the membrane, it is “bound” to the liposome membrane. These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly reduces liposome uptake by the macrophage-mononuclear cell system (“MMS”) and the reticuloendothelial system (“RES”). For example, as described in US Pat. No. 4,920,016, the entire disclosure of which is hereby incorporated by reference. Liposomes modified with opsonization-inhibiting moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes referred to as “stealth” liposomes.

  Stealth liposomes are known to accumulate in tissues fattened by porous or “leaky” microvasculature. Thus, target tissues characterized by such microvasculature defects, such as solid tumors, will efficiently accumulate these liposomes. See Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. Furthermore, the reduced uptake by RES reduces the toxicity of stealth liposomes by preventing significant accumulation in the liver and spleen. Therefore, the liposome of the present invention modified with an opsonization-inhibiting moiety can deliver the double-stranded molecule of the present invention to tumor cells.

  Opsonization-inhibiting moieties suitable for liposome modification are preferably water-soluble polymers having a molecular weight of from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives such as methoxy PEG or PPG and stearic acid PEG or PPG; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear , Branched or dendrimeric polyamidoamines; polyacrylic acid; polyalcohols such as polyvinyl alcohol and polyxylitol chemically linked carboxy or amino groups, and ganglioside GM. sub. 1. And gangliosides. Also suitable are copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof. Further, the opsonization-inhibiting polymer can be a block copolymer of PEG and any of polyamino acids, polysaccharides, polyamidoamines, polyethyleneamines, or polynucleotides. Opsonization-inhibiting polymers are natural polysaccharides containing amino acids or carboxylic acids, such as galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharide or oligo Sugars (linear or branched); or carboxylated polysaccharides or oligosaccharides, such as those reacted with a derivative of a carboxylic acid having a resulting carboxy group linkage.

Preferably, the opsonization inhibiting moiety is PEG, PPG, or a derivative thereof. Liposomes modified with PEG or PEG derivatives are sometimes referred to as “PEGylated liposomes”.
The opsonization-inhibiting moiety can be bound to the liposome membrane by any of a number of well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipophilic anchor and then bound to a membrane. Similarly, dextran polymer is a stearylamine lipophilic anchor via reductive amination using a solvent mixture such as tetrahydrofuran and water in a ratio of Na (CN) BH ₃ and 30:12 at 60 ° C. It can be derivatized.

  Vectors expressing the double-stranded molecules of the invention have been described above. Such a vector expressing at least one double-stranded molecule of the present invention comprises a Milus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations ( Administration can be with or directly with a suitable delivery reagent including, for example, polylysine) or liposomes. Methods for delivering a recombinant viral vector that expresses a double-stranded molecule of the invention to a region of cancer in a subject are within the skill of the artisan.

  The double-stranded molecule of the invention can be administered to a subject by any means suitable for delivering the double-stranded molecule to a cancer site. For example, double-stranded molecules can be administered by gene gun, electroporation, or by other suitable parenteral or enteral routes of administration. Suitable enteral routes of administration include oral, rectal or intranasal delivery.

  Suitable parenteral routes of administration include intravascular administration (eg, intravenous bolus injection, intravenous drip, intraarterial bolus injection, intraarterial drip and catheter drop injection into the vasculature); For example, peritumoral and intratumoral injection); subcutaneous injection or deposition including subcutaneous infusion (such as by an osmotic pump); direct application to the vicinity of the cancer or region of the site, eg, by catheter or other indwelling device One (eg, a suppository or implant containing a porous, non-porous, or gelatinous material); and inhalation. The injection or infusion of the double-stranded molecule or vector is preferably given at or near the cancer site.

  The double-stranded molecules of the present invention can be administered in a single dose or multiple doses. When administration of the double-stranded molecule of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. The direct injection of the substance into the tissue is preferably at or near the cancer site. Multiple injections of substances into the cancer site or nearby tissue are particularly preferred.

  One skilled in the art can also readily determine an appropriate dosing regimen for administering a double-stranded molecule of the invention to a given subject. For example, the double-stranded molecule can be administered to the subject at once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule can be administered to the subject once or twice daily for a period of about 3 to about 28 days, more preferably about 7 to about 10 days. In a preferred dosing regimen, the double-stranded molecule is infused once a day for 7 days at or near the site of the cancer. Where the dosage regimen includes multiple administrations, it is understood that an effective amount of a double-stranded molecule administered to a subject can include the total amount of double-stranded molecules administered throughout the dosage regimen.

In the context of the present invention, a cancer that overexpresses the SUV39H2 gene can be treated with at least one active ingredient selected from the group consisting of:
(A) the double-stranded molecule of the present invention,
(B) DNA encoding it, and (c) a vector encoding it.
Cancers to be treated include, but are not limited to, lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor.

Therefore, before administration of the double-stranded molecule of the present invention as an active ingredient, it is confirmed whether the expression level of the SUV39H2 gene in the cancer cell or tissue to be treated is enhanced compared to normal cells of the same organ. It is preferable. Accordingly, in one embodiment, the present invention provides a method for treating a cancer that (over) expresses the SUV39H2 gene, which method may comprise the following steps:
i) determining the expression level of the SUV39H2 gene in a cancer cell or tissue obtained from a subject having a cancer to be treated;
ii) comparing the expression level of the SUV39H2 gene with a normal control; and
iii) at least one component selected from the group consisting of:
(A) the double-stranded molecule of the present invention,
(B) DNA encoding the double-stranded molecule, and (c) a vector encoding the double-stranded molecule,
In a subject having cancer that overexpresses the SUV39H2 gene compared to a normal control. Alternatively, the present invention provides at least one component selected from the group consisting of:
(A) the double-stranded molecule of the present invention,
(B) DNA encoding the double-stranded molecule, and (c) a vector encoding the double-stranded molecule,
A pharmaceutical composition for use in administration to a subject having cancer that overexpresses the SUV39H2 gene is also provided. In other words, the present invention includes the following:
(A) the double-stranded molecule of the present invention,
(B) DNA encoding the double-stranded molecule, or (c) a vector encoding the double-stranded molecule,
A method for identifying a subject to be treated with, wherein the method may comprise determining the expression level of the SUV39H2 gene in a subject-derived cancer cell or tissue, wherein An increase in the level compared to the normal control level of the gene indicates that the subject has a cancer that may be treated with the double-stranded molecule of the invention.

The cancer treatment method of the present invention is described in further detail below.
The subject to be treated by the method of the present invention is preferably a mammal. Exemplary mammals include, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.
According to the present invention, the expression level of the SUV39H2 gene in a cancer cell or tissue obtained from a subject is determined. This expression level can be determined at the transcription (nucleic acid) product level using methods known in the art. For example, hybridization methods (eg, Northern hybridization), chips or arrays, probes, RT-PCR can be used to determine the transcript level of the SUV39H2 gene.

Alternatively, translation products may be detected for the treatment of the present invention.
For example, the amount of observed protein (SEQ ID NO: 22, 24, 26, 28 or 30) may be determined.
As another method for detecting the expression level of the SUV39H2 gene based on the translation product, the staining intensity may be measured via immunohistochemical analysis using an antibody against the SUV39H2 protein. Specifically, in this measurement, intense staining indicates an increased presence / level of protein and at the same time a high expression level of the SUV39H2 gene.
The method for detecting or measuring either or both of the SUV39H2 polypeptide or the polynucleotide encoding the same can be exemplified as described above (cancer diagnosis method).

Composition containing the double-stranded molecule of the present invention:
In addition to the above, the present invention also provides a pharmaceutical composition comprising the double-stranded molecule of the present invention or a vector encoding this molecule.
In the context of the present invention, the term “composition” refers to a product that contains a specific component in a specific amount, as well as any product that results directly or indirectly from a combination of a specific amount of a specific component. Used to refer to Such terms, when used with respect to the modifier “medicament” (as in “pharmaceutical composition”), a product containing an active ingredient and any inactive ingredients that form a carrier, and Any directly or indirectly resulting from any combination of two or more components, complex formation or aggregation, or from the dissociation of one or more components, or from other types of reactions or interactions of one or more components Are intended to encompass products comprising: Thus, in the context of the present invention, the term “pharmaceutical composition” refers to any product made by mixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier. .

  The phrase “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein includes, but is not limited to, liquid or solid fillers, diluents, additives, solvents or encapsulating substances. It means, without limitation, a pharmaceutically or physiologically acceptable material, composition, substance or excipient.

  The term “active ingredient” as used herein refers to a substance in a composition that is biologically or physiologically active. In particular, in the context of pharmaceutical compositions, the term “active ingredient” refers to a substance that exhibits an objective pharmaceutical effect. For example, in the case of a pharmaceutical composition for use in the treatment or prevention of cancer, the active ingredient in the drug or composition is at least one biological or physiological for cancer cells and / or tissues. The effect may be brought about directly or indirectly. Preferably, such effects may include reducing or inhibiting cancer cell proliferation, killing cancer cells and / or tissues, and the like. Prior to formulation, the “active ingredient” can be referred to as “bulk”, “drug substance” or “technical product”.

Of particular interest to the present invention are the following compositions [1]-[20]:
[1] A composition for inhibiting the growth of cancer cells and / or for the treatment and prevention of cancer, wherein the cancer cells and cancer express the SUV39H2 gene and against the SUV39H2 gene An isolated double-stranded molecule or a vector encoding the double-stranded molecule, wherein the double-stranded molecule inhibits SUV39H2 gene expression and cell proliferation, wherein the double-stranded molecule is A composition composed of a sense strand and an antisense strand complementary thereto, and hybridizes to each other to form a double-stranded molecule;
[2] The composition according to [1] above, wherein the double-stranded molecule acts on mRNA matching the target sequence of SEQ ID NO: 19 or 20.
[3] The composition according to [1], wherein the double-stranded molecule includes a sequence whose sense strand corresponds to the target sequence of SEQ ID NO: 19 or 20.
[4] The cancer according to any one of [1] to [3], wherein the cancer to be treated is selected from lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer, and soft tissue tumor. A composition of;
[5] The composition according to any one of [1] to [4], wherein the double-stranded molecule is less than about 100 nucleotides in length;
[6] The composition according to [5] above, wherein the double-stranded molecule is less than about 75 nucleotides in length;
[7] The composition according to [6] above, wherein the double-stranded molecule is less than about 50 nucleotides in length;
[8] The composition according to [7] above, wherein the double-stranded molecule is less than about 25 nucleotides in length;
[9] The composition according to [8] above, wherein the double-stranded molecule is about 19 to about 25 nucleotides in length;
[10] The any one of [1] to [9] above, wherein the double-stranded molecule is composed of a single polynucleotide comprising a sense strand and an antisense strand linked by an intervening single strand. A composition of;
[11] The double-stranded molecule has the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′ Wherein [A] is a sense strand sequence containing a sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, [B] is an intervening single strand consisting of 3 to 23 nucleotides, and [A ′] is the antisense strand comprising a sequence complementary to the target sequence, the composition according to [10] above;
[12] The composition according to any one of [1] to [11], wherein the double-stranded molecule is composed of RNA;
[13] The composition according to any one of [1] to [11], wherein the double-stranded molecule is composed of both DNA and RNA;
[14] The composition according to [13] above, wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[15] The composition according to [14] above, wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[16] The composition according to [13] above, wherein the double-stranded molecule is a chimera of DNA and RNA;
[17] Both the region adjacent to the 3'-end of the antisense strand, or the region adjacent to the 5'-end of the sense strand and the region adjacent to the 3'-end of the antisense strand are composed of RNA. The composition according to [14] above;
[18] The composition according to [17] above, wherein the adjacent region is composed of 9 to 13 nucleotides;
[19] The composition according to any one of the above [1] to [18], wherein the double-stranded molecule comprises a 3 ′ overhang; and [20] the composition is a transfection enhancing agent and pharmaceutically The composition according to any one of the above [1] to [19], which contains an acceptable carrier.

The compositions of the present invention are described in further detail below. The double-stranded molecules of the invention are preferably formulated prior to administration to a subject according to techniques known in the art. The pharmaceutical composition of the present invention is at least aseptic and pyrogen-free.
As used herein, “pharmaceutical composition” includes formulations for human and veterinary use.

  In the context of the present invention, suitable pharmaceutical formulations of the present invention include oral, rectal, nasal, topical (including buccal, sublingual, and transdermal), vaginal or parenteral (intramuscular, subcutaneous). And suitable formulations for administration (including intravenous) or for administration by inhalation or insufflation. Other formulations include implantable devices that release therapeutic agents and patch patches. If desired, the above formulations may be adapted to provide sustained release of the active ingredient. Methods for preparing the pharmaceutical compositions of the present invention are within the scope of techniques known in the art and are described, for example, in “Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985)”. The entire disclosure of which is hereby incorporated by reference.

  The pharmaceutical composition of the present invention comprises a double-stranded molecule of the present invention or a vector encoding the same (for example, 0.1 to 90% by weight), mixed with a physiologically acceptable carrier medium, or Containing physiologically acceptable salts. Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

  According to the present invention, the composition may comprise multiple types of double-stranded molecules, each molecule may be directed to the same target sequence of SUV39H2 and directed to different target sequences. May be. For example, the composition may include a double-stranded molecule directed to the SUV39H2 gene or gene product. Alternatively, for example, the composition may comprise a double-stranded molecule directed to one, two or more target sequences of the SUV39H2 gene.

Furthermore, the composition of the invention may comprise a vector encoding one or more double-stranded molecules. For example, the vector may encode one, two or several types of double-stranded molecules of the invention. Alternatively, the composition of the present invention may contain a plurality of types of vectors each encoding a different double-stranded molecule.
Moreover, the double-stranded molecule of the present invention may be contained as a liposome in the composition of the present invention. For details of liposomes, refer to the section “Methods of treating cancer using double-stranded molecules”.

The pharmaceutical composition of the present invention may also contain conventional pharmaceutical additives and / or additives. Examples of suitable pharmaceutical additives include stabilizers, antioxidants, osmotic pressure adjusting agents, buffering agents, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (eg, tromethamine hydrochloride), addition of chelating agents (eg, such as DTPA or DTPA-bisamide) or calcium chelate complexes (eg, DTPA). Calcium, CaNaDTPA-bisamide) or, optionally, addition of calcium or sodium salts (eg calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). The pharmaceutical compositions of the invention can be packaged for use in liquid form or can be lyophilized.
For solid compositions, conventional non-toxic solid carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. can be used. .

  For example, a solid pharmaceutical composition for oral administration comprises any of the carriers and excipients listed above and 10-95%, preferably 25-75% of one or more of the present invention. Chain molecules can be included. A pharmaceutical composition for aerosol (inhalation) administration comprises 0.01-20% by weight, preferably 1-10% by weight of one or more of the inventions encapsulated in a liposome as described above. Double-stranded molecules, and propellants can be included. A carrier can also be included as desired, such as lecithin for intranasal delivery.

  In addition to the above, the composition of the present invention may contain other pharmaceutically active ingredients as long as they do not inhibit the in vivo function of the double-stranded molecule of the present invention. For example, the composition may include a chemotherapeutic agent conventionally used for the treatment of cancer. The pharmaceutical composition may also contain other active ingredients such as antimicrobial agents, immunosuppressive agents or preservatives. Furthermore, in addition to the ingredients specifically mentioned above, it should be understood that the formulations of the present invention may contain other agents customary in the art for the type of formulation in question. It is. For example, those suitable for oral administration may contain a fragrance.

In another aspect, the invention relates to the use of a double-stranded nucleic acid molecule of the invention or a vector encoding a double-stranded nucleic acid molecule of the invention in the manufacture of a pharmaceutical composition for the treatment of cancer characterized by the expression of the SUV39H2 gene Also provide. For example, the present invention is the use of a double-stranded nucleic acid molecule that inhibits the expression of the SUV39H2 gene in a cell for the manufacture of a pharmaceutical composition for treating a cancer that expresses the SUV39H2 gene, A molecule comprising a sense strand and an antisense strand complementary thereto and hybridizing to each other to form a double-stranded nucleic acid molecule, targeting the sequence of SEQ ID NO: 19 or 20, or a double-stranded nucleic acid molecule It relates to the use of vectors.
The present invention further provides a double-stranded nucleic acid molecule of the present invention or a vector encoding a double-stranded nucleic acid molecule for use in the treatment of a cancer that expresses the SUV39H2 gene.

  The present invention relates to a method or process for the manufacture of a pharmaceutical composition for one or both of the treatment and prevention of cancer characterized by the expression of SUV39H2 gene, said method or process comprising Formulating a double stranded nucleic acid molecule that inhibits expression of the SUV39H2 gene or a vector encoding the double stranded nucleic acid molecule and a pharmaceutically or physiologically acceptable carrier, wherein the cell overexpresses the gene The molecule comprises a sense strand and a complementary antisense strand, hybridizes to each other to form a double-stranded nucleic acid molecule, and targets the sequence of SEQ ID NO: 19 or 20. Provide further.

In another aspect, the present invention also provides a method or process for the manufacture of a pharmaceutical composition for the treatment and / or prevention of cancer characterized by expression of the SUV39H2 gene, wherein The method or process comprises mixing the active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule or two that inhibits the expression of the SUV39H2 gene in the cell. A vector encoding a single stranded nucleic acid molecule, which overexpresses this gene, which contains a sense strand and an antisense strand complementary thereto, and hybridizes to each other to form a double stranded nucleic acid molecule. Target the sequence or vector of SEQ ID NO: 19 or 20.
In the following, the present invention will now be described in more detail with reference to examples. However, the following materials, methods and examples are only intended to illustrate aspects of the present invention and are not intended to limit the scope of the invention in any way. Accordingly, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Example

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: General Methods Tissue Sample and RNA Preparation Bladder tissue sample and RNA preparation were as previously described [Wallard MJ, et al,
Br J Cancer 2006; 94: 569-577]. Briefly, 125 primary urothelial cancer surgical specimens were collected and snap frozen in liquid nitrogen through cystectomy or transurethral resection (TURBT) of bladder tumors. Twenty-eight specimens of normal bladder urothelial tissue were collected from areas of macroscopically normal bladder urothelium in patients with no evidence of malignancy. Vimentin was expressed primarily in cells derived from the mesenchymal system and was used as a stromal marker. Uroplakin is a urothelial differentiation marker and is conserved in up to 90% of epithelial derived tumors [Olsburgh J et al, J Pathol 2003; 199: 41-49]. The use of the tissue for this study was approved by the Cambridgeshire Local Research Ethics Committee (Ref 03/018).

Cell Culture The cancer cell lines used in this study were as follows: lung adenocarcinoma (ADC) NCI-H1781, NCI-H1373, LC319, A549 and PC-14; lung squamous cell carcinoma (SCC) SK -MES-1, NCI-H2170, NCI-H520, NCI-H1703 and RERF-LC-AI; large cell lung cancer (LCC) LX1; small cell lung cancer (SCLC) SBC-3, SBC-5, DMS273 and DMS114; Bladder cancer SW780, RT4 and SCaBER. Human small airway epithelial cells (SAEC), normal human fibroblasts (IMR-90, WI-38 and CCD-18Co) were used as normal control cells. All cell lines were grown in monolayers in appropriate medium supplemented with 10% fetal bovine serum and 1% antibiotic / antifungal solution (Sigma). All cells were maintained at 37 ° C. in humidified air with 5% CO ₂ (all cell lines except SW780) or without CO ₂ (SW780). Cells were transfected with FuGENE6 (ROCHE, Basel, Switzerland) according to the manufacturer's protocol.

Expression profiling in cancer using cDNA microarrays A genome-wide cDNA microarray was established using 36,864 cDNAs selected from the UniGene database of the National Center for Biotechnology Information (NCBI). The microarray system was constructed essentially as previously described [Kitahara O et al, Cancer Res 2001; 61: 3544-3549]. Briefly, cDNAs were amplified by RT-PCR using poly (A) ⁺ RNA isolated from various human organs as a template; amplicon lengths ranged from 200 to 1100 bp and were repetitive No sequence or poly (A) sequence was included. Many types of tumors and corresponding non-tumor tissues were prepared in 8 micrometer sections as previously described [Kitahara O et al, Cancer Res 2001; 61: 3544-3549]. A total of 30,000-40,000 individual cancer or non-cancerous cells are selectively recovered using the EZ cut system (SL MicroTest GmbH, Germany) according to the manufacturer's protocol. It was. Total RNA extraction, T7-based amplification, and probe labeling were performed as previously described [Kitahara O et al, Cancer Res 2001; 61: 3544-3549]. A 2.5 microgram quantitative measure (A measure) of double amplified RNA (aRNA) from each cancerous and non-cancerous tissue was then labeled with Cy3-dCTP or Cy5-dCTP, respectively. .

Quantitative real-time PCR
As previously described, 125 bladder cancers and 28 normal bladder tissues were prepared at Cambridge Addenbrooke's Hospital. Specific primers for all of GAPDH (housekeeping gene), SDH (housekeeping gene) and SUV39H2 were designed for quantitative RT-PCR reactions (primer sequences in Table 1) [Yoshimatsu M et al, Int J Cancer 2011; 128: 562-573]. PCR reactions were performed using the LightCycler® 480 system (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol. 50% SYBR GREEN universal PCR master mix without UNG (Applied Biosystem, Warrington, UK), 50 nM forward and reverse primers each, and 2 microliters of reverse transcribed cDNA were applied. Amplification conditions were 45 ° C. for 5 minutes, then 95 ° C. for 10 seconds, 55 ° C. for 1 minute, and 72 ° C. for 10 seconds, respectively. The reaction was then heated at 95 ° C. for 15 seconds and 65 ° C. for 1 minute to generate a melting curve and cooled at 50 ° C. for 10 seconds. Reaction conditions for target gene amplification were as described above, and 5 ng of reverse transcribed RNA equivalent was used for each reaction. mRNA levels were normalized to GAPDH and SDH expression.

Immunohistochemistry Immunohistochemical analysis was performed using a specific polyclonal rabbit SUV39H2 antibody produced by SIGMA GENOSYS as previously described [Takawa M, et al. Cancer Sci 2011; 102 : 1298-305, Toyokawa G, et al. Neoplasia 2011; 13: 887-98, Toyokawa G, et al. Mol Cancer 2011; 10:65]. For all tissue samples, the EnVision + kit / horseradish peroxidase (Dako, Glostrup, Denmark) was applied. Briefly, paraffin-embedded tumor specimen slides were treated with high pH 9 (S2367, Dako Cytomation, Carpinteria, CA, USA) in antigen recovery solution under high pressure (125 ° C., 30 seconds) and peroxidase Treated with blocking reagent and then with protein blocking reagent (K130, X0909, Dako Cytomation). Tissue sections were incubated with rabbit anti-SUV39H2 polyclonal antibody followed by HRP labeled secondary antibody (Dako Cytomation). Antigen was visualized using substrate chromogen (Dako liquid DAB chromogen; Dako Cytomation). Finally, tissue samples were stained with Mayer's hematoxylin (Muto pure chemicals Ltd, Tokyo, Japan, Hematoxylin QS, Vector Laboratories) for 20 seconds to distinguish nuclei from cytoplasm. Since the staining intensity within each tumor tissue core was almost uniform, the intensity of SUV39H2 staining was evaluated semi-quantitatively according to the following criteria: negative (no recognizable staining in tumor cells) and positive (tumor) Recognizable brown staining in more than 30% of cell nuclei).

Immunocytochemistry HeLa cells were transfected with pCAGGS-n3FC-SUV39H2 (3 × FLAG-SUV39H2). 48 hours after transfection, cultured cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 minutes at room temperature and 0.5% Triton X-100 in PBS (Sigma ). Fixed cells were blocked with 5% BSA in PBS for 1 hour and incubated overnight at 4 ° C. with monoclonal mouse FLAG antibody. They were then incubated with Alexa Fluor 594 conjugated secondary antibody (Molecular Probes) and Alexa Fluor 488 phalloidin (Molecular Probes). Nuclei were stained with 4 ′, 6-diamidino-2-phenylindole (DAPI). Stained cells were observed using a Leica confocal microscope.

siRNA transfection siRNA oligonucleotide duplexes were purchased from SIGMA Genosys to target the human SUV39H2 transcript. siEGFP, siFFLuc and siNegative Control (siNC), which is a mixture of three different oligonucleotide duplexes, were used as control siRNAs. These siRNA sequences are listed in Table 2. siRNA duplexes (100 nM final concentration) were transfected into lung and bladder cancer cell lines using Lipofectamine 2000 (Invitrogen) for 72 hours and cell viability was cell counting as previously described. Tested using the Kit-8 (Cell Counting Kit-8) (Dojindo, Kumamoto, Japan) and colony formation assay [Sato N et al, Cancer Res 2010; 70: 5326-5336].

Flow cytometry assay (FACS)
SW780 and A549 cells were treated with siSUV39H2 (siSUV39H2 # 1 and siSUV39H2 # 2) or control siRNA (siEGFP and siNC) and cultured for 72 hours at 37 ° C. in a CO ₂ incubator. A quantification of 1 × 10 ⁵ cells was collected by trypsinization and stained with propidium iodide (PI) according to manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). Cells were analyzed for detailed cell cycle status by FACScan (BECKMAN COULTER, Brea, Calif.) Equipped with “MultiCycle for Windows” software (BECKMAN COULTER). The percentage of cells in the G ₀ / G ₁ , S and G ₂ / M phase of the cell cycle was determined from at least 20,000 ungated cells.

Coupled Cell Cycle and Cell Proliferation Assay Using the 5′-bromo-2′-deoxyuridine (BrdU) flow kit (BD Pharmingen, San Diego, Calif.), Cell cycle kinetics were determined and proliferating cells BrdU incorporation into DNA was measured. The assay was performed according to the manufacturer's protocol. Briefly, MEFs (1 × 10 ⁵ per well) were seeded in 6-well tissue culture plates for 72 hours, followed by addition of 10 micromolar BrdU and incubation continued for another 30 minutes. Both floating and adherent cells were pooled from triplicate wells per treatment point, fixed in a solution containing paraformaldehyde and detergent saponin, and incubated with DNAse for 1 hour at 37 ° C (30 micron). Grams / sample). FITC-labeled anti-BrdU antibody (1:50 dilution, in wash buffer; BD Pharmingen, San Diego, Calif.) Was added and incubation continued at room temperature for 20 minutes. Cells are washed with wash buffer and total DNA is stained with 7-amino-actinomycin D (7-AAD; 20 microliters / sample) followed by flow cytometric analysis using FACScan (BECKMAN COULTER). The total DNA content (7-AAD) was determined with CXP analysis software Ver. 2.2 (BECKMAN COULTER).

Microarray hybridization and statistical analysis for downstream gene elucidation Microarray analysis and gene ontology pathway analysis to identify downstream genes were performed as previously described [Yoshimatsu M et al, Int J Cancer 2011; 128 : 562-573]. Purified total RNA was labeled and hybridized on an Affymetrix “Gene Chip U133 Plus 2.0” oligonucleotide array (Affymetrix, Santa Clara, Calif.) According to the manufacturer's instructions. Probe signal intensity was normalized by RNA and quantile (using R and Bioconductor). Path analysis is performed using a hypergeometric test, which calculates the up / down-regulated gene set and the probability of overlap between each GO category compared to another randomly sampled gene list. Tests for identified up / down-regulated genes to test whether they are significantly enriched in each category of “biological processes” (857 categories) defined by the gene ontology database Applied (FDR <= 0.05).

Example 2: Abnormal expression of SUV39H2 in clinical lung and bladder cancer tissues To analyze the expression level of SUV39H2 in clinical samples, 16 normal and 14 lung cancer tissues (9 NSCLC and 5 SCLC) were used. qRT-PCR was performed and found that SUV39H2 was significantly upregulated in lung cancer tissue relative to normal tissue (FIGS. 1A and 1B). Of the normal tissues, only the testis showed a high SUV39H2 expression level comparable to that of lung cancer tissue. Immunohistochemical analysis was performed to assess the protein expression level of SUV39H2, and no significant staining was observed in some normal tissues except the testis (FIG. 1C). Subsequent tissue microarray analysis showed that SUV39H2 stained positively in 34 of 64 lung cancer cases (53.1%; FIG. 2 and Table 3) and no significant staining was observed in normal living organs (FIG. 2C). These results imply that SUV39H2 is overexpressed with high frequency in lung cancer tissues, while its expression in normal tissues including living organs appears to be very low. To analyze the association between SUV39H2 expression and clinical outcome, we performed a tumor tissue microarray on an array containing 328 archived NSCLC cases (FIG. 10). SUV39H2 stained positive in 217 cases (66.16%) and negative in 111 cases (33.84%). On the other hand, no significant statistical significance was observed between SUV39H2 positive and patient characteristics (Table 11).

  Next, SUV39H2 expression levels were analyzed in clinical bladder tissue by qRT-PCR (British) and a significant increase in SUV39H2 was found in cancer tissue compared to non-tumor bladder tissue (P <0.05, Mann-Whitney U test; FIG. 3A). No significant differences were identified by tumor subclassification according to tumor grade, metastasis status, gender, recurrence status and smoking history (Table 4). SUV39H2 overexpression was also identified by cDNA expression analysis of Japanese bladder clinical cases (FIG. 3B). SUV39H2 protein expression levels in bladder tissue were then examined by immunohistochemistry and strong SUV39H2 staining was observed in the nucleus of cancer tissue, but no significant staining was observed in non-tumor tissue (FIG. 3C). . Tissue microarray analysis showed that overexpression of SUV39H2 was observed in 16 of 29 bladder cancer cases (55.2%; Table 5). In addition, microarray expression analysis using a large number of clinical samples showed that SUV39H2 expression was significantly upregulated in various types of cancer in addition to lung and bladder cancer (Table 6). This data suggests that elevated expression of SUV39H2 is frequently observed in human carcinogenesis.

Example 3: SUV39H2 is important for cancer cell growth The role of SUV39H2 in cancer cell growth was investigated. qRT-PCR was performed to measure the expression level of SUV39H2 in various cell lines at the RNA level, confirming elevated SUV39H2 expression in lung and bladder cancer cells compared to non-cancerous cells (FIG. 4A and FIG. 6). Overexpression of SUV39H2 protein in cancer cells was confirmed by Western blotting (FIG. 4B). To test the effect of SUV39H2 knockdown on cancer cell growth, two independent siRNAs targeting SUV39H2 were prepared and each of them was transfected into cancer cells. The inventors then confirmed significant suppression of SUV39H2 expression after SUV39H2 siRNA treatment compared to control siRNA (siEGFP and siNC; FIG. 4C). Cell proliferation assays performed on cancer cells transfected with these siRNAs revealed significant growth inhibition in lung cancer cell line A549 and bladder cancer cell line SW780 (FIG. 4D). These siRNAs expressed undetectable levels of SUV39H2 (FIG. 4A) when transfected into normal cell line CCD-18Co cells, they had no effect on proliferation (FIG. 4D). This implies that the suppression of cancer cell growth by treatment with these specific siRNAs does not provide the mechanism used by normal cells and thus directly affects the growth mechanism specific to cancer cells. . Suppression of cancer cell growth by siRNA targeting SUV39H2 was confirmed by colony formation assay (FIGS. 4E and 7A). In order to test whether SUV39H2 has carcinogenic activity, the inventor performed a clonogenic potential assay. Wild type SUV39H2 (SUV39H2 Wt) vector and enzyme inactivated SUV39H2 (SUV39H2 delta-SET) vector were transfected into COS-7 cells with mock vector as a control. A clonogenicity assay was performed for each culture (FIG. 4F). Cells transfected with the wild-type SUV39H2 vector form more colonies than cells transfected with the enzyme-inactivated SUV39H2 vector or mock control vector, and it is therefore SUV39H2 methylation that promotes carcinogenesis in the cells. Active. Since SUV39H2 is overexpressed early in cancer progression, SUV39H2 may play a critical role in human carcinogenesis. To further evaluate the mechanism of growth inhibition induced by siRNA, FACS analysis was performed to examine the detailed cell cycle status of cancer cells. The ratio of cancer cells in G ₁ phase, while was significantly higher in cells treated with siSUV39H2 compared to cells treated with control siRNA, the ratio of S phase cells was significantly reduced (Fig. 5) . These data indicate that SUV39H2 plays a critical role in cancer cell growth regulation, particularly in the G ₁ / S transition.

Example 4: Identification of downstream genes of SUV39H2 by microarray expression analysis In order to identify how SUV39H2 can contribute to human carcinogenesis, the inventors sought pathways and downstream genes associated with SUV39H2. After 24 hours of SUV39H2 siRNA treatment in SW780 and A549 cells, microarray hybridization analysis was performed as previously described [Yoshimatsu M, et al,
Int J Cancer 2011; 128: 562-573]. Expression profiles were analyzed by Affymetrix “HG-U133 Plus 2.0” array and compared to profiles from cells treated with control siRNA (siEGFP and siFFLuc). The data showed that 146 genes were down-regulated after SUV39H2 knockdown, while 64 genes were up-regulated: these 210 genes were considered downstream genes regulated by SUV39H2 inhibition ( FIG. 8, Table 7 and Table 8). Signal path analysis of downstream candidates from the gene ontology database suggested that SUV39H2 regulates pathways for cell cycle regulation and chromatin modification (Table 9). Importantly, mass spectrometry analysis identified proteins involved in cell cycle regulation and chromatin function as SUV39H2 interaction partners (Table 10). Thus, SUV39H2 deregulation is likely to contribute to human carcinogenesis, partly through the regulation of histone and chromatin functions.

Example 5: Screening for inhibitors of SUV39H2 methyltransferase activity GST-fused SUV39H2 (SEQ ID NO: 33; GST: amino acid positions 1-98, SUV39H2 fragment: amino acid positions 99-410) is a 0.35 mM biotinylated histone H3 peptide ( SEQ ID NO: 31), 0.1 mM S- adenosyl-L-[methyl ^- 3 H] with methionine and the test substance, methyltransferase buffer (20 mM _{tris-HCl, 50mM NaCl, 10mM MgCl 2,} 0.03% Tween-80 And 10 mM DTT) at room temperature for 1 hour in a volume of 20 microliters. After 1 hour incubation at room temperature, the reaction was stopped by adding 20 microliters of scintillation proximity assay (SPA) bead buffer containing 60 micrograms of streptavidin-coated PVT beads, and then the light emitted from the beads was Measurements were made using a scintillation counter.

  As a result of the evaluation of a number of chemically synthesized compounds by the above-described assay, several compounds were identified that inhibit the methyltransferase activity of SUV39H2.

DISCUSSION Epigenetic modifications, including DNA methylation, covalent histone modifications, nucleosome positioning and miRNAs, play important roles in normal mammalian development and regulation of gene expression [Sharma S et al, Carcinogenesis 2010; 31: 27-36]. In addition to the significance of epigenetics in normal physiological function, its deregulation has been linked to cancer [Sharma S et al, Carcinogenesis 2010; 31: 27-36], cardiovascular disease [Turunen MP et al, Biochim Biophys Acta 2009; 1790: 886-891], metabolic diseases [Symonds ME et al. Nat Rev Endocrinol 2009; 5: 604-610], and autoimmune diseases [Javierre BM et al. Genome Res 2010; 20: 170-179 It is deeply involved in various types of diseases such as Among epigenetic changes, aberrant histone modifications are involved in the development and progression of cancer [Chi P et al. Nat Rev Cancer 2010; 10: 457-469, Portela A et al. Nat Biotechnol 2010; 28: 1057-1068]. The association of acetylation / deacetylation kinetics with human carcinogenesis has been studied most extensively among histone modifications [Cortez CC et al, Mutat Res 2008; 647: 44-51], but the growing body of evidence is Show that histone methyltransferase is also important for carcinogenesis [Schneider R et al. Trends Biochem Sci 2002; 27: 396-402].

  With the exception of Dot1 / DOT1L, all identified histone lysine methyltransferases (HKMTs) are SET [Su (var) 3-9, Enhancer-of-zeste, Trithorax] domain [Jenuwein T et al. Cell Mol Life Sci. 1998; 54: 80-93] carry a conserved methyltransferase domain named. Among HKMTs, the Su (var) gene was originally identified by genetic screening for centromeric positional effects in Drosophila melanogaster and fission yeast (Shizosaccharomyces pombe) [Reuter Get al. Bioessays 1992; 14: 605- 612, Allshire RC et al. Genes Dev 1995; 9: 218-233], a characterized family member actually represents an enzyme capable of modifying chromatin [Wallrath LL Curr Opin Genet Dev 1998; 8: 147 −153]. Subsequently, Suv39h2, a mouse homologue of human SUV39H2, was isolated and characterized as a second methyltransferase that targets H3K9 and contributes to heterochromatin formation [O'Carroll D et al. Mol Cell Biol. 2000; 20 : 9423-9433]. Importantly, subsequent studies suggested that Suv39-depleted mice phenotype impaired survival, chromosomal instability and spermatogenesis [Peters AH et al. Cell 2001; 107: 323- 337].

  In addition, another study showed that inhibition of Suv39 expression resulted in reduced di- and trimethylation of H3K9 at telomeres, more concomitant H3K9 monomethylation and reduced chromobox protein binding. [Garcia-Cao M, et al. Nat Genet 2004; 36: 94-99]. Although these data imply epigenetic regulation by Suv39h2 on diverse mammalian biological functions, their role in human carcinogenesis remains to be elucidated.

As revealed herein, the inventors found significant upregulation of SUV39H2 in lung and bladder cancer tissues by qPCR along with microarray-based expression profiles of numerous clinical tissues compared to normal tissues SUV39H2 is abnormally overexpressed in various types of cancer. Subsequently, SUV39H2 was elucidated to play an important role in regulating cancer cell growth, inhibition of SUV39H2 expression resulted in growth inhibition of lung and bladder cancer cell lines, cell number decreased in S phase, G It was confirmed that it increased in the _first phase (FIGS. 4 and 5). Path analysis of cells treated with SUV39H2 siRNA indicated that SUV39H2 may regulate multiple biological pathways including chromatin modifications. This result is consistent with our findings that SUV39H2 regulates the expression of numerous proteins associated with histone modifications and chromatin dynamics and actually interacts directly with them (Table 6).

The importance of SUV39H2 in the progression to S cell proliferation and _{G 1} has been confirmed in Suv39hDN (SUV39H1 / SUV39H2 double null) mouse embryonic fibroblasts (MEFs, FIGS. 7B and 7C). Again, microarray and gene ontology analysis showed that SUV39H2 could be associated with multiple biological processes including the cell cycle (data not shown). Recently, it has been reported that LSD1 can demethylate MYPT1 and this process may be involved in human carcinogenesis [Cho HS, et al. Cancer Res. 2010]. In addition to the inventor's work, a series of reports show that diverse non-histone proteins are subject to methylation / demethylation cycles and that this process is a transcriptional activity, protein-level stability, and interaction partner [Huang J, et al. Nature 2006; 444: 629-632, Huang J et al. Nature 2007; 449: 105-108, Esteve PO et al. Proc Natl Acad Sci USA 2009; 106: 5076-5081, Guo Z et al. Nat Chem Biol 2010; 6: 766-773]. Given that SUV39H2 may interact with a number of non-histone proteins (Table 10), further studies should be conducted to identify new substrates for SUV39H2.

These expression analyzes indicate that the expression level of SUV39H2 in various types of cancer tissues is significantly increased compared to normal tissues (FIGS. 1, 2 and 3) and that siSUV39H2 significantly suppresses cancer cell proliferation. (FIGS. 4C-4E). Since the expression level of SUV39H2 in various types of normal tissues is significantly lower (FIG. 1C and FIG. 9: data from BioGPS), this gene appears to be an ideal target for cancer therapy. The fact that inhibitors targeting DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) have been approved for hematological malignancies is specific to epigenetic mechanisms for cancer therapy More and more attention is drawn to the development of inhibitors [Cortez CC et al. Mutat Res 2008; 647: 44-51, Ma WW et al. CA Cancer J Clin 2009; 59: 111-137]. Importantly, structural analysis of human H3K9 methyltransferase was performed and provided important insights to guide the development of selective inhibitors of H3K9 methyltransferase [Wu H, et al. PLoS One 2010; 5: e8570]. Taking into account these findings, it seems feasible to develop inhibitors of SUV39H2 for cancer therapy. Since the development of inhibitors targeting methyltransferases has just begun [Copeland RA et al. Nat Rev Drug Discov 2009; 8: 724-732], further validation of the function of this enzyme will be in the near future. It may ensure the usefulness of this approach.
Industrial applicability

  The cancer gene expression analysis described here uses a combination of laser-capture dissection and genome-wide cDNA microarrays to target specific genes as targets for cancer prevention and therapy. Identified. Based on the expression of this differentially expressed gene, SUV39H2, the present invention provides a novel molecular diagnostic marker for cancer identification and detection. Thus, the present invention also provides a novel diagnostic strategy using SUV39H2.

Furthermore, as described herein, SUV39H2 is involved in cancer cell survival. Thus, the present invention also provides novel molecular targets for cancer treatment and prevention. They can be useful in the development of new therapeutic and prophylactic agents without side effects.
The inventor has shown that cell proliferation is suppressed by double-stranded molecules that specifically target the SUV39H2 gene. Therefore, the double-stranded molecule for the SUV39H2 gene is useful for the development of anticancer drugs.

Moreover, substances that block the expression of SUV39H2 protein or inhibit its activity are anti-cancer agents, particularly anti-cancer agents for the treatment of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer or soft tissue tumor. The therapeutic utility as a cancer drug can be found.
The methods described herein are also useful for the identification of additional molecular targets for cancer prevention, diagnosis and treatment. The data provided here increases the overall understanding of cancer, facilitates the development of new diagnostic strategies, and provides clues for the identification of molecular targets for therapeutic and prophylactic agents. Such information contributes to a deeper understanding of tumorigenesis and provides an indicator for developing new strategies for cancer diagnosis, treatment, and ultimately prevention.

Although the invention has been described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. Will. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims (29)

A method for detecting or diagnosing cancer in a subject or determining a predisposition to developing cancer, the method comprising the step of determining the expression level of the SUV39H2 gene in a biological sample from the subject. Wherein an increase in the level relative to a normal control level of the gene indicates that the subject is suffering from or at risk of developing cancer, wherein the expression level is A method determined by a method selected from the group consisting of:
(A) detecting the mRNA of the SUV39H2 gene;
(B) detecting a protein encoded by the SUV39H2 gene, and (c) detecting a biological activity of the protein encoded by the SUV39H2 gene.   The method of claim 1, wherein the increase is at least 10% higher than the normal control level.   The method of claim 1, wherein the biological sample from the subject comprises a biopsy specimen, saliva, sputum, blood, serum, plasma, pleural effusion or urine. A kit for detecting or diagnosing cancer or determining a predisposition to develop cancer, comprising a reagent selected from the group consisting of:
(A) a reagent for detecting mRNA of the SUV39H2 gene;
(B) a reagent for detecting the protein encoded by the SUV39H2 gene, and (c) a reagent for detecting the biological activity of the protein encoded by the SUV39H2 gene.   The kit according to claim 4, wherein the reagent is a probe for a transcript of the SUV39H2 gene.   The kit according to claim 4, wherein the reagent is an antibody against a protein encoded by the SUV39H2 gene.   The method according to any one of claims 1 to 3, or the kit according to any one of claims 4 to 6, wherein the biological activity to be detected is a cell growth promoting activity or a methyltransferase activity. .   The method or claim 1 according to any one of claims 1 to 3, wherein the cancer is selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor. The kit according to any one of 4 to 6. A method for screening candidate substances for the inhibition or inhibition of cancer cell growth or treatment and / or prevention of cancer, comprising the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting a binding activity between the polypeptide or a functional equivalent thereof and the test substance; and (c) selecting a test substance that binds to the polypeptide or a functional equivalent thereof. A method for screening candidate substances for the inhibition or inhibition of cancer cell growth or treatment and / or prevention of cancer, comprising the following steps:
(A) contacting the test substance with cells expressing the SUV39H2 gene; and (b) selecting a test substance that reduces the expression level of the SUV39H2 gene compared to the expression level in the absence of the test substance. . A method for screening candidate substances for the inhibition or inhibition of cancer cell growth or treatment and / or prevention of cancer, comprising the following steps:
(A) contacting the test substance with a SUV39H2 polypeptide or a functional equivalent thereof;
(B) detecting the biological activity of the polypeptide of step (a) or a functional equivalent thereof; and (c) comparing the biological activity detected in the absence of said test substance Selecting a test substance that inhibits the biological activity of the polypeptide or functional equivalent thereof.   The method according to claim 11, wherein the biological activity is a cell growth promoting activity or a methyltransferase activity. A method for screening candidate substances for the inhibition or inhibition of cancer cell growth or treatment and / or prevention of cancer, comprising the following steps:
a) contacting a test substance with a cell into which a vector containing a transcription regulatory region of the SUV39H2 gene and a reporter gene expressed under the control of the transcription regulatory region has been introduced;
b) measuring the expression or activity level of the reporter gene; and
c) selecting a test substance that reduces the expression or activity level of the reporter gene compared to the level in the absence of the test substance. A method for screening candidate substances for the inhibition or inhibition of cancer cell growth or treatment and / or prevention of cancer, comprising the following steps:
(A) contacting the test substance with a cell expressing a SUV39H2 gene and a downstream gene selected from the genes shown in Tables 7 and 8;
(B) detecting the expression level of the downstream gene, and (c) selecting a test substance that changes the expression level of the downstream gene compared to the expression level detected in the absence of the test substance.   The downstream gene of the SUV39H2 gene is selected from the genes shown in Table 7, and step (c) is compared to the detected expression level detected in the absence of the test substance. 15. The method according to claim 14, which is a step of selecting a test substance that increases the expression level.   The downstream gene of the SUV39H2 gene is selected from the genes shown in Table 8, and step (c) reduces the expression level of the downstream gene compared to the expression level detected in the absence of the test substance The method according to claim 14, which is a step of selecting a test substance to be performed.   The method according to any one of claims 9 to 16, wherein the cancer is selected from the group consisting of lung cancer, cervical cancer, bladder cancer, esophageal cancer, osteosarcoma, prostate cancer and soft tissue tumor.   A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, and the antisense strand comprises the target Comprising a nucleotide sequence complementary to the sequence, the sense strand and the antisense strand hybridize to each other to form a double-stranded molecule, wherein the double-stranded molecule expresses the SUV39H2 gene A double-stranded molecule that, when introduced into a cell, inhibits the expression of the SUV39H2 gene.   The double-stranded molecule of claim 18, wherein the double-stranded molecule is about 19 to about 25 nucleotides in length.   19. The double stranded molecule of claim 18, wherein the double stranded molecule is a single polynucleotide molecule comprising a sense strand and an antisense strand linked via a single stranded nucleotide sequence. The double-stranded molecule is represented by the general formula 5 ′-[A]-[B]-[A ′]-3 ′ or 5 ′-[A ′]-[B]-[A] -3 ′.
Wherein [A] is a sense strand comprising a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 19 or 20, and [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; 21. The double-stranded molecule according to claim 20, wherein and [A ′] are antisense strands comprising a nucleotide sequence complementary to the target sequence.   A vector encoding the double-stranded molecule according to any one of claims 18 to 21.   A vector comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises the nucleotide sequence of SEQ ID NO: 19 or 20, and the antisense strand nucleic acid is complementary to the sense strand A vector comprising a nucleotide sequence, wherein the transcript of the sense strand and the antisense strand hybridizes to each other to form a double-stranded molecule, and the vector inhibits the expression of the SUV39H2 gene.   One or both methods of treating and preventing cancer in a subject, wherein the method encodes the subject with a pharmaceutically effective amount of a double-stranded molecule against the SUV39H2 gene or the double-stranded molecule. Administering a vector, wherein said double-stranded molecule inhibits SUV39H2 gene expression as well as cell proliferation when introduced into a cell expressing the SUV39H2 gene.   The method according to claim 24, wherein the double-stranded molecule is the double-stranded molecule according to any one of claims 18 to 21.   25. The method of claim 24, wherein the vector is the vector of claim 22 or 23.   A composition for one or both of the treatment and prevention of cancer, said composition comprising a pharmaceutically effective amount of a double-stranded molecule against the SUV39H2 gene or a vector encoding said double-stranded molecule and pharmaceutically A composition comprising an acceptable carrier, wherein the double-stranded molecule inhibits SUV39H2 gene expression as well as cell proliferation when introduced into a cell expressing the SUV39H2 gene.   The composition according to claim 27, wherein the double-stranded molecule is the double-stranded molecule according to any one of claims 18 to 21. 28. The composition of claim 27, wherein the vector is the vector of claim 22 or 23.